Methods for production of platelets from pluripotent stem cells and compositions thereof

ABSTRACT

Methods for production of platelets from pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are provided. These methods may be performed without forming embryoid bodies or clusters of pluripotent stem cells, and may be performed without the use of stromal inducer cells. Additionally, the yield and/or purity can be greater than has been reported for prior methods of producing platelets from pluripotent stem cells. Also provided are compositions and pharmaceutical preparations comprising platelets, preferably produced from pluripotent stem cells.

RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationSer. No. PCT/US2013/077334, filed Dec. 21, 2013, and claims the benefitof U.S. Provisional Application Ser. No. 61/740,699, filed Dec. 21,2012, and U.S. Provisional Application Ser. No. 61/787,476, filed Mar.15, 2013, all entitled “METHODS FOR PRODUCTION OF PLATELETS FROMPLURIPOTENT STEM CELLS AND COMPOSITIONS THEREOF”, the contents of all ofwhich are incorporated by reference herein in their entirety.

BACKGROUND OF INVENTION

Platelets are tiny blood cells that perform the vital and highlyspecialized function of blood clotting. Almost a trillion plateletscirculate in the average person's blood, and the turnover is such thatthe entire platelet population is replaced every 10 days. Thisrepresents a tremendous amount of ongoing platelet production. Plateletshave a highly organized cytoskeleton and intracellular stores of over300 proteins, which they secrete at sites of blood vessel injury.Platelets also play a role in inflammation, blood vessel growth, andtumor metastasis.

After vascular injury, platelets rapidly adhere to damaged blood vesselsand trigger a complex cascade of events that result in thrombusformation. The demand for platelet transfusions has continued toincrease during the last several decades (51). Using conventionalmethods, platelets can only be stored for less than a week, creating acontinuous challenge for donor-dependent programs. Shortages in thesupply of platelets can have potentially life-threatening consequences,especially in patients where multiple transfusions are necessary.Repeated transfusions may also lead to refractory responses that arelinked to immunity mediated host reaction and may require costly patientmatching (52; 53). The ability to generate platelets in vitro,particularly patient-matched platelets, would provide significantadvantages in these clinical scenarios.

Limitations in the supply of platelets can have potentiallylife-threatening consequences for transfusion-dependent patients withunusual/rare blood types, particularly those who are alloimmunized, andpatients with cancer or leukemia who, as often happens, develop plateletalloimmunity. Frequent transfusion of platelets is clinically necessaryin these patients because the half-life of transfused human platelets is4-5 days. Moreover, platelets from volunteer donor programs are at theconstant risk of contaminations by various pathogens. Platelets cannotbe stored frozen using conventional techniques, thus the ability togenerate platelets in vitro would provide significant advances forplatelet replacement therapy in clinical settings.

For more than a decade, human hematopoietic stem cells (HSC, CD34+) frombone marrow (BM), cord blood (CB) or peripheral blood (PB) have beenstudied for megakaryocyte (MK) and platelet generation. Using certaincombinations of cytokines, growth factors and/or stromal feeder cells,functional platelets have been produced from HSCs with significantsuccess (1; 2). However, HSCs are still collected from donors and havelimited expansion capacity under current culture conditions, whichinterferes with large-scale production and future clinical applications.

Human embryonic stem cells (hESC) can be propagated and expanded invitro indefinitely, providing a potentially inexhaustible and donorlesssource of cells for human therapy. Differentiation of hESCs intohematopoietic cells in vitro has been extensively investigated for thepast decade. The directed hematopoietic differentiation of hESCs hasbeen successfully achieved in vitro by means of two different types ofculture systems. One of these employs co-cultures of hESCs with stromalfeeder cells, in serum-containing medium (3; 4). The second type ofprocedure employs suspension culture conditions in ultra-low cellbinding plates, in the presence of cytokines with/without serum (5-7);its endpoint is the formation of cell aggregates or embryoid bodies(“EBs”). Hematopoietic precursors as well as mature, functionalprogenies representing erythroid, myeloid, macrophage, megakaryocyticand lymphoid lineages have been identified in both of the abovedifferentiating hESC culture systems (3-6:8-14). Previous studies alsogenerated megakaryocytes/platelets from hESCs by co-culturing withstromal cells in the presence of serum (15; 16). However, the yield ofmegakaryocytes/platelets in the above-described studies was low (15;16).

SUMMARY OF INVENTION

The present disclosure provides methods for production of platelets frompluripotent stem cells, such as human embryonic stem cells (hESCs) andinduced pluripotent stem cells (iPSCs or iPS cells), such as humaninduced pluripotent stem cells (hiPSCs or hiPS cells). These methods maybe performed without forming embryoid bodies, and may be performedwithout the use of stromal inducer cells. Additionally, the yield and/orpurity can be greater than has been reported for prior methods ofproducing platelets from pluripotent stem cells. Because platelets maybe produced with greater efficiency and on a larger scale, the methodsand compositions of the present disclosure have great potential for usein medicinal transfusion purposes. Additionally, because platelets donot have a nucleus and contain only minimal genetic material,preparations of the present disclosure may be irradiated beforetransfusion to effectively eliminate any contaminating nucleated cells,such as an undifferentiated hESC. Therefore, possible presence ofnucleated cells should not present a safety issue.

Platelets collected from donors have very limited shelf life and areincreasingly needed for prophylactic transfusions in patients. Incontrast to donor dependent cord blood or bone marrow CD34+ humanhematopoietic stem cells, human embryonic stem cells (hESCs) can be apromising alternative source for continuous in vitro production ofplatelets under controlled conditions. As further described herein, thedisclosure provides systems and methods to generate megakaryocytes (MKs)from pluripotent stem cells under serum- and stromal-free conditions. Inexemplary embodiments, pluripotent stem cells are directed towardsmegakaryocytes through differentiation of hemogenic endothelial cells(PVE-HE, which are further described below). A transient multi-potentialcell population expressing CD31, CD144, and CD105 markers has beenidentified at the end of PVE-HE culture. In the presence of TPO, SCF andother cytokines in feeder-free and serum-free suspension culture, up to100 fold expansion can be achieved from hESCs or hiPS cells to MKs in18-20 days. Such methods can provide robust in vitro generation of MKsfrom pluripotent stem cells. When cultured under feeder-free conditions,pluripotent stem cell-derived MKs may be used to generate platelet-likeparticles (platelet or platelet-like particle produced from humaninduced pluripotent stem cells (hiPSC-PLTs) or from human embryonic stemcells (ES PLTs)). These hiPSC-PLTs and ES-PLTs are responsive tothrombin stimulation and able to participate in micro-aggregateformation.

In one aspect, the disclosure provides a pharmaceutical preparation thatis suitable for use in a human patient comprising at least 10⁸platelets.

Additionally, the disclosure provides a pharmaceutical preparationcomprising platelets differentiated from human stem cells, e.g., atleast 10⁸ platelets. Optionally, the preparation may be substantiallyfree of leukocytes. Optionally, substantially all of the platelets maybe functional.

The pharmaceutical preparation may comprise 10⁹-10¹⁴ platelets,optionally 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ platelets.

The platelets may have one or more of the following attributes: a meanplatelet volume range of 9.7-12.8 fL; a unimodal distribution of size inthe preparation; and/or a log normal platelet volume distributionwherein one standard deviation is less than 2 μm³ (preferably less than1.5 μm³, 1 μm³ or even 0.5 μm³).

The platelets may be positive for at least one of the following markers:CD41a and CD42b.

The platelets may be human platelets.

At least 50%, 60%, 70%, 80% or 90% of the platelets may be functional,and optionally may be functional for at least 2, 3 or 4 days afterstorage at room temperature.

In another aspect, the disclosure provides a bioreactor having weaklyadherent or non-adherent megakaryocytes that produce functionalplatelets without feeder cells.

In another aspect, the disclosure provides a composition comprising atleast 10⁹ megakaryocyte lineage specific progenitors (MLPs).

In another aspect, the disclosure provides a cryopreserved compositioncomprising MLPs.

In another aspect, the disclosure provides a bank comprisingcryopreserved MLPs.

The MLPs may be of defined HLA types.

The cryopreserved composition may be HLA matched to a patient.

In another aspect, the disclosure provides a cryopreserved compositionor bank comprising 10⁹ to 10¹⁴ MLPs, optionally 10⁹, 10¹⁰, 10¹¹, 10¹²,10¹³ or 10¹⁴ MLPs.

In another aspect, the disclosure provides a method for producingplatelets from megakaryocytes or MPLs comprising the steps of: (a)providing a non-adherent culture of megakaryocytes; (b) contacting themegakaryocytes or MPLs with TPO or a TPO agonist to cause the formationof proplatelets in culture, wherein the proplatelets release platelets;and (c) isolating the platelets.

In another aspect, the disclosure provides a method for producingplatelets from megakaryocytes or MPLs comprising the steps of: (a)providing a non-adherent culture of megakaryocytes or MPLs; (b)contacting the megakaryocytes or MPLs with hematopoietic expansionmedium and optionally (1) TPO or a TPO agonist, SCF, IL-6 and IL-9 or(2) TPO or a TPO agonist, SCF, and IL-11, which may cause the formationof pro-platelets in culture, wherein the pro-platelets releaseplatelets. Said method may further comprise (c) isolating the platelets.

The TPO agonist comprises one or more of: ADP, epinephrine, thrombin,collagen, TPO-R agonists, TPO mimetics, second-generation thrombopoieticagents, romiplostim, eltrombopag (SB497115, Promacta), recombinant humanthrombopoietin (TPO), pegylated recombinant human megakaryocyte growthand development factor (PEG-rHuMGDF), Fab 59, AMG 531, Peg-TPOmp, TPOnonpeptide mimetics, AKR-501, monoclonal TPO agonist antibodies,polyclonal TPO agonist antibodies, TPO minibodies, VB22B sc(Fv)2, domainsubclass-converted TPO agonist antibodies, MA01G4G344, recombinant humanthrombopoietins, recombinant TPO fusion proteins, or TPO nonpeptidemimetics.

Optionally, substantially all the isolated platelets may be functional.

The non-adherent culture of megakaryocytes or MPLs may be a feeder-freeculture.

The culture in step (b) may be in a medium comprising one or more of:Stem Cell Factor (SCF) at 0.5-100 ng/ml, Thrombopoietin (TPO) at 10-100ng/ml, and Interleukin-11 (IL-11) at 10-100 ng/ml, at least one ROCKinhibitor, and/or Heparin at 2.5-25 Units/ml.

The culture in step (b) may be in a medium comprising one or more of:TPO at 10-100 ng/ml, SCF at 0.5-100 ng/ml, IL-6 at 5-25 ng/ml, IL-9 at5-25 ng/ml, at least one ROCK inhibitor, and/or Heparin at 2.5-25units/ml.

The at least one ROCK inhibitor may comprise Y27632, which Y27632 may bein a concentration of 2-20 μM, about 3-10 μM, about 4-6 μM or about 5μM.

The method may further comprise subjecting the megakaryocytes to ashearing force.

At least 2, 3, 4, or 5 platelets per megakaryocyte may be produced.

At least 50 platelets per megakaryocyte may be produced.

At least 100, 500, 1000, 2000, 5000, or 10000 platelets permegakaryocyte may be produced.

At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, orat least 95% of said platelets may be CD41a+ and/or CD42b+, e.g., CD41a+and CD42b+.

The platelets may be produced in the absence of feeder cells, and/or maybe produced in the absence of stromal feeder cells.

The platelets may be produced in the absence of any xenogeneic cells.

The platelets may be human.

The megakaryocytes or MPLs may be cultured in the presence of anexogenously added protease inhibitor. Said megakaryocytes or MPLs may becultured in the presence of an exogenously added MMP inhibitor. Saidmegakaryocytes or MPLs may be cultured in the presence of an exogenouslyadded MMP8 inhibitor. Said megakaryocytes or MPLs may be cultured in thepresence of an exogenously added MMP8 specific inhibitor and a pan MMPinhibitor.

The megakaryocytes or MPLs may be cultured at a temperature of about 39°C.

The megakaryocytes or MPLs may be generated by steps comprising: (a)culturing pluripotent stem cells to form hemogenic endothelial cells(PVE-HE); (b) culturing the hemogenic endothelial cells to form MLPs;and optionally (c) culturing the MLPs to form megakaryocytes. Thepluripotent stem cells may be human.

The hemogenic endothelial cells may be cultured in step (b) in thepresence of a c-myc inhibitor, such as a BET inhibitor. Said BETinhibitor may be IBET151.

The hemogenic endothelial cells may be derived without embryoid bodyformation.

The pluripotent stem cells may be induced pluripotent stem cells (iPSC).The iPSC may be human.

The hemogenic endothelial cells may be derived without embryoid bodyformation.

The hemogenic endothelial cells may be differentiated from thepluripotent stem cells under low oxygen conditions comprising 1% to 10%oxygen, 2% to 8% oxygen, 3% to 7% oxygen, 4% to 6% oxygen, or about 5%oxygen.

The megakaryocytes may be differentiated from the MLPs at a temperaturebetween 38-40 degrees C., or about 39 degrees C.

In another aspect, the disclosure provides a pharmaceutical preparationcomprising platelets produced by any of the method described herein,e.g., any of the methods above.

The preparation may be suitable for use in a human patient. For example,the preparation may be suitable for use in a human patient andsubstantially free of leukocytes. Said preparation may comprise at least10⁸ platelets.

In a further aspect the disclosure provides the use of a compositioncomprising platelets (e.g., a composition as described herein, such asin the preceding paragraphs) or a composition comprising plateletsproduced by a method as described herein (e.g., a method described inthe preceding paragraphs) in the manufacture of a medicament for thetreatment of a patient in need thereof or suffering from a disease ordisorder affecting clotting or a disease or disorder treatable thereby.

The disease or disorder may comprise thrombocytopenia, trauma, ablood-borne parasite, or malaria.

In another aspect, the disclosure provides a method of treating apatient in need of platelet transfusion, comprising administering acomposition comprising platelets (e.g., a composition as describedherein, such as in the preceding paragraphs) or a composition comprisingplatelets produced by a method as described herein (e.g., a methoddescribed in the preceding paragraphs) to said patient.

The method may be effective to treat a disease or disorder comprisingthrombocytopenia, trauma, a blood-borne parasite, or malaria.

In another aspect, the present disclosure provides a compositioncomprising an isolated PVE-HE cell, which is optionally derived from apluripotent stem cell, and which is optionally produced according to themethods described herein. The PVE-HE composition can be a preparation ofcells highly enriched for PVE-HE cells, i.e., may be at least 70%(cellular composition) PVE-HE cells, or even at least 75%, 80%, 85%,90%, 95% or even 98% PVE-HE cells. In certain embodiments, the PVE-HEcomposition includes a suitable cryopreservative, and the compositionmay be frozen/cryopreserved such as for transport or storage.

In one aspect, the present disclosure provides a composition comprisingan isolated iPS-PVE-HE cell, which is optionally produced according tothe methods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated hES-PVE-HE, which is optionally produced according to themethods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated PVE-HE-MLP which is optionally derived from a pluripotentstem cell and which is optionally produced according to the methodsdescribed herein. The PVE-HE-MLP composition can be a preparation ofcells highly enriched for PVE-HE-MLP cells, i.e., may be at least 70%(cellular composition) PVE-HE-MLP cells, or even at least 75%, 80%, 85%,90%, 95% or even 98% PVE-HE-MLP cells. In certain embodiments, thePVE-HE-MLP composition includes a suitable cryopreservative, and thecomposition may be frozen/cryopreserved such as for transport orstorage.

In one aspect, the present disclosure provides a composition comprisingan isolated iPS-PVE-HE-MLP, which is optionally produced according tothe methods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated hES-PVE-HE-MLP, which is optionally produced according tothe methods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated PVE-HE-MLP-MK which is optionally derived from a pluripotentstem cell, and which is optionally produced according to the methodsdescribed herein. The PVE-HE-MLP-MK composition can be a preparation ofcells highly enriched for PVE-HE-MLP-MK cells, i.e., may be at least 70%(cellular composition) PVE-HE-MLP-MK cells, or even at least 75%, 80%,85%, 90%, 95% or even 98% PVE-HE-MLP-MK cells. In certain embodiments,the PVE-HE-MLP-MK composition includes a suitable cryopreservative, andthe composition may be frozen/cryopreserved such as for transport orstorage

In one aspect, the present disclosure provides a composition comprisingan isolated iPS-PVE-HE-MLP-MK, which is optionally produced according tothe methods described herein.

In one aspect, the present disclosure provides a composition comprisingan isolated hES-PVE-HE-MLP-MK, which is optionally produced according tothe methods described herein.

In another aspect, the disclosure provides a pharmaceutical preparationthat is suitable for use in a human patient comprising at least 10⁸platelets, wherein the preparation is substantially free of leukocytesand wherein substantially all of the platelets are functional.

In various embodiments, the pharmaceutical preparations are irradiatedin order to remove or inactivate nucleated cells.

In another aspect, the disclosure provides a pharmaceutical preparationthat is suitable for use in a human patient comprising at least 10⁸functional platelets, wherein the mean plasma half-life of thefunctional platelets in the preparation is at least four days.

The pharmaceutical preparation of may comprise 10⁹-10¹⁴ platelets,optionally 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ platelets.

The pharmaceutical preparation may comprise platelets having one or moreof the following attributes: a mean platelet volume range of 9.7-12.8fL; a unimodal distribution of size in the preparation; and/or a lognormal platelet volume distribution wherein one standard deviation isless than 2 μm³ (preferably less than 1.5 μm³, 1 μm³ or even 0.5 μm³).

The pharmaceutical preparation may comprise platelets that are positivefor at least one of the following markers: CD41a and CD41b.

At least half of the platelets may be functional for at least two,three, four or five days after storage at room temperature e.g. (22-25°C.). For example, at least 60%, 70%, 80% or 90% may be functional for atleast two days. The platelets may be stored at room temperature for atleast five days.

In another aspect, the disclosure provides a cryopreserved bank orpreparation of MLPs.

MLPs may be collected from the PVE-HE when they start to float up insuspension. Preferably the MLPs are not plated and preferably the MLPsare not allowed to adhere, thereby avoiding differentiation into othercell types and facilitating production of MKs.

The bank or preparation may comprise 10⁹ to 10¹⁴ MLPs, optionally 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ MLPs.

One MLP may yield at least 2,3, 4, 5, or more platelets. In exemplaryembodiments, a composition of 6×10¹⁰ to 1.2×10¹¹ MLPs is providedsufficient to generate a therapeutic dose of 300-600×10⁹ platelets at ayield of 5 platelets per MLP. In exemplary embodiments, a composition of3-6×10⁹ MLPs is provided sufficient to generate a therapeutic dose at ayield of at least 100 platelets per MLP.

In another aspect, the disclosure provides a bioreactor having weaklyadherent or non-adherent megakaryocytes that produce functionalplatelets without feeder cells. Weakly adherent cells, including saidmegakaryocytes, can be mechanically separated from each other or from asurface, for example, by washing with mild force using a serologicalpipette. Preferably the MKs are cultured under non-adherent conditions,though immobilized by the structural features of the bioreactor (e.g.,pores, channels, gates, fibers or the like) which is thought to promotemaintenance of MK phenotypes. The MKs are disposed in the bioreactorsuch that shear forces may be applied to the MK culture to improve theefficiency of platelet production. Shear forces may be applied toimmobilized MKs, to illustrate, by the flow of culture media, such thatproplatelet processes of the MKs extend into the flow/stream of culturemedia which aids in the release of platelets from those extendingprocesses. For example, a microfluidic chamber or chip may be used tocontrol shear stress, which may increase the platelet yield per MK. Inone aspect, MKs can be seeded into one channel of a microfluidic chipand media can be flowed past the MKs at near physiologic rates. Incertain embodiments, the microfluidic platelet bioreactor recapitulatesfeatures such as bone marrow stiffness, extracellular matrixcomposition, micro-channel size, and blood flow stability to makefunctional human platelets. In certain embodiments, the bioreactorcreates a two-dimensional flow culture system that is a biomimetic foran artificial blood vessel system. To further illustrate, the bioreactorcan be composed of (e.g., biodegradable) scaffolds with ordered arraysof pores made to mimic in vivo bone marrow. Within the system, two flowsin different directions in which the angle between the directions offlow is less than 90 degrees (e.g., about 60 degrees) can providesuitable pressure and shear stress to MKs to promote plateletgeneration. The significance to the bioreactor design can be measured byits increased efficiency and/or scalability, as well as its ultimateability to generate platelets from pluripotent stem cells with intactintegrin αIIbβ3 activation responses after agonist stimulation (i.e.,such platelets are functional).

In another aspect, the disclosure provides a composition comprising atleast 10⁹ MLPs

In another aspect, the disclosure provides a cryopreserved compositioncomprising MLPs.

In another aspect, the disclosure provides a method for producingplatelets from megakaryocytes comprising the steps of: a) providing anon-adherent culture of megakaryocytes; b) contacting the megakaryocyteswith TPO or TPO agonists to cause the formation of proplatelets inculture, wherein the proplatelets release platelets; and c) isolatingthe platelets.

Thrombopoietin (TPO) is thought to be a key cytokine involved inthrombopoiesis and megakaryopoiesis, and is the endogenous ligand forthe thrombopoietin receptor that is expressed on the surface ofplatelets, megakaryocytes, and megakaryocytic precursors. TPO is a332-amino acid (95 kDa) glycoprotein that contains 2 domains: areceptor-binding domain (residues 1-153) and a carbohydrate-rich domain(residues 154-332) that is highly glycosylated and is important forprotein stability. The TPO receptor, c-Mpl (also known as CD110), is atypical hematopoietic cytokine receptor and contains 2 cytokine receptorhomology modules. TPO binds only the distal cytokine receptor homologymodule and thereby initiates signal transduction. In the absence of thedistal cytokine receptor homology module, c-Mpl becomes active,suggesting that the distal cytokine receptor homology module functionsas an inhibitor of c-Mpl until it is bound by TPO. Binding by TPOactivates Janus kinase 2 (Jak2) signal transducers and activators oftranscription (STAT) signaling pathway to drive cell proliferation anddifferentiation. Megakaryocyte growth and development factor (MGDF) isanother thrombopoietic growth factor. Recombinant forms of TPO and MGDF,including human and pegylated forms, may be used to induce megakaryocyteand platelet differentiation and maturation. TPO receptor-activatingpeptides and fusion proteins (i.e. Fab 59, romiplostim/AMG 531, orpegylated (Peg-TPOmp)) may be used in place of TPO. Nonpeptide mimetics(Eltrombopag (SB497115, Promacta), and AKR-501) bind and activate theTPO receptor by a mechanism different from TPO and may have an additiveeffect to TPO. TPO agonist antibodies (i.e. MA01G4G344) or minibodies(i.e. VB22B sc(Fv) 2) that activate the TPO receptor may also be used tomimic the effect of TPO. Exemplary TPO agonists are disclosed in Stasiet al., Blood Reviews 24 (2010) 179-190, and Kuter, Blood. 2007;109:4607-4616 which is each hereby incorporated by reference in itsentirety. Exemplary TPO agonists include: ADP, epinephrine, thrombin andcollagen, and other compounds identified in the literature as TPO-Ragonists or TPO mimetics, second-generation thrombopoietic agents,Romiplostim, Eltrombopag (SB497115, Promacta), first-generationthrombopoietic growth factors, recombinant human thrombopoietin (TPO),pegylated recombinant human megakaryocyte growth and development factor(PEG-rHuMGDF), TPO peptide mimetics, TPO receptor-activating peptidesinserted into complementarity-determining regions of Fab (Fab 59), AMG531 (a “peptibody” composed of 2 disulphide-bonded human IgG1-HCconstant regions (an Fc fragment) each of which is covalently bound atresidue 228 with 2 identical peptide sequences linked via polyglycine),Peg-TPOmp (a pegylated TPO peptide agonist), orally available TPOagonists, TPO nonpeptide mimetics, AKR-501, monoclonal TPO agonistantibodies, polyclonal TPO agonist antibodies, TPO minibodies such asVB22B sc(Fv)2, domain subclass-converted TPO agonist antibodies such asMA01G4G344, Recombinant human thrombopoietins, or Recombinant TPO fusionproteins, TPO nonpeptide mimetics. Wherever TPO is used as an embodimentof the invention, exemplary TPO agonists can be substituted for TPO infurther embodiments of the invention.

In another aspect, the disclosure provides a method for producingplatelets from megakaryocytes comprising the steps of a) providing anon-adherent culture of megakaryocytes or megakaryocyte progenitors, b)contacting the megakaryocytes or megakaryocyte progenitors with acomposition comprising hematopoietic expansion media to cause theformation of proplatelets in culture, wherein the proplatelets releaseplatelets, and c) isolating the platelets.

In exemplary embodiments, substantially all the isolated platelets arefunctional. In exemplary embodiments, the non-adherent culture ofmegakaryocytes or megakaryocyte progenitors is a feeder-free cultureand/or is free of xenogeneic cells. Accordingly, the disclosure providesmethods for generating platelets without feeder cells.

The culture in step (b) may be performed in a medium comprising one ormore of Stem Cell Factor (SCF), Thrombopoietin (TPO), Interleukin-11(IL-11), a ROCK Inhibitor such as Y27632 and/or Heparin. The culture instep (b) may be in a medium comprising one or more of TPO, SCF, IL-6,IL-9, a ROCK Inhibitor such as Y27632, and/or Heparin.

In one embodiment, the hematopoietic expansion medium comprisesStemSpam™ ACF (ACF) (available from StemCell Technologies Inc.), and mayfurther comprise TPO (thrombopoietin) or TPO agonist, SCF (Stem CellFactor), IL-6 (interleukin 6) and IL-9 (interleukin 9), which may beprovided as StemSpam™ CC220 cytokine cocktail (CC220) (available fromStemCell Technologies Inc.). It may optionally comprise a ROCK inhibitorand/or Heparin. TPO, SCF, IL-6, IL-9, and IL-11 are known megakaryocytedevelopment and maturation factors (Stasi et al., Blood Reviews 24(2010) 179-190).

In one embodiment, the hematopoietic expansion medium comprisesStemline-II Hematopoietic Stem Cell Expansion Medium (Stemline-II)(available from Sigma Aldrich), and may further comprise TPO or TPOagonist, SCF, and IL-11. It may optionally comprise a ROCK inhibitorand/or Heparin. The ROCK inhibitor may be but is not limited to Y27632.

In another embodiment, the hematopoietic expansion medium comprisesIscove's Modified Dulbecco's Medium (IMDM) as a basal medium, humanserum albumin (recombinant or purified), iron-saturated transferrin,insulin, b-mercaptoethanol, soluble low-density lipoprotein (LDL), andcholesterol (which may be referred to herein as a defined componentmedium), and may further comprise TPO or TPO agonist, SCF, and IL-11. Itmay optionally comprise a ROCK inhibitor and/or Heparin. The ROCKinhibitor may be but is not limited to Y27632.

In another embodiment, the hematopoietic expansion medium comprisesIscove's Modified Dulbecco's Medium (IMDM) as a basal medium, humanserum albumin (recombinant or purified), iron-saturated transferrin,insulin, b-mercaptoethanol, soluble low-density lipoprotein (LDL), andcholesterol (which may be referred to herein as a defined componentmedium), and may further comprise TPO (thrombopoietin) or TPO agonist,SCF (Stem Cell Factor), IL-6 (interleukin 6) and IL-9 (interleukin 9).It may optionally comprise a ROCK inhibitor and/or Heparin.

The culture in step (b) may be performed in a medium comprising ACF,Stemline-II or the defined component medium of the preceding paragraph,as well as (1) one or more of SCF (e.g., at 0.5-100 ng/ml), TPO (e.g.,at 10-100 ng/ml), IL-6 (e.g., at 5-25 ng/ml), IL-9 (e.g., at 5-25 ng/ml)and Heparin (e.g., at 2.5-25 Units/ml); (2) one or more of TPO (e.g., at10-100 ng/ml), SCF (e.g., at 0.5-100 ng/ml), IL-6 (e.g., at 5-25 ng/ml),IL-9 (e.g., at 5-25 ng/ml), Y27632 (e.g., at 5 μM, or optionally 2-20μM, or optionally an effective concentration of another ROCK inhibitor),and Heparin (e.g., at 0.5-25 units/ml); (3) one or more of TPO (e.g., at10-100 ng/ml), SCF (e.g., at 0.5-100 ng/ml), IL-11 (e.g., at 5-25ng/ml), Y27632 (e.g., at 5 μM or optionally 2-20 μM, or optionally aneffective concentration of another ROCK inhibitor), and Heparin (e.g.,at 2.5-25 Units/ml); or (4) one or more of TPO (e.g., at 10-100 ng/ml),SCF (e.g., at 0.5-100 ng/ml), IL-6 (e.g., at 5-25 ng/ml), IL-9 (e.g., at5-25 ng/ml), Y27632 (e.g., at 5 μM or optionally 2-20 μM, or optionallyan effective concentration of another ROCK inhibitor), and Heparin(e.g., at 2.5-25 Units/ml).

The method may further comprise subjecting the megakaryocytes to ashearing force.

The method may yield at least 2, 3, 4, or 5 platelets per megakaryocyte,at least 20, 30, 40 or 50 platelets per megakaryocyte, or at least 100,500, 1000, 2000, 5000, or 10000 platelets per megakaryocyte.

At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, orat least 95% of said platelets may be CD41a+ and CD42b+.

The platelets may be produced in the absence of feeder cells or stromalfeeder cells.

The platelets may be produced in the absence of any xenogeneic cells.

The platelets may be human.

The platelets may be CD41a+ and/or CD42b+.

In another aspect, the disclosure provides methods of producingmegakaryocyte progenitors (also referred to herein as MLPs) (such asthose utilized in a method of producing platelets or for anotherpurpose) which may be generated by the steps of (a) culturingpluripotent stem cells to form hemogenic endothelial cells (PVE-HE); and(b) culturing the hemogenic endothelial cells to form megakaryocytesprogenitors (MLPs). Step (a) may be carried out in the presence of ananimal-component free medium comprised of Iscove's Modified Dulbecco'sMedium (IMDM), human serum albumin, iron-saturated transferrin, insulin,b-mercaptoethanol, soluble low-density lipoprotein (LDL), cholesterol,bone morphogenetic protein 4 (BMP4) (e.g., at 50 ng/ml), basicfibroblast growth factor (bFGF) (e.g., at 50 ng/ml), and vascularendothelial growth factor (VEGF) (e.g., at 50 ng/ml). Step (b) may becarried out in Iscove's modified Dulbecco's medium (IMDM), Ham's F-12nutrient mixture, Albucult (rh Albumin), Polyvinylalcohol (PVA),Linoleic acid, SyntheChol (synthetic cholesterol), Monothioglycerol(a-MTG), rh Insulin-transferrin-selenium-ethanolamine solution,protein-free hybridoma mixture II (PFHMII), ascorbic acid 2 phosphate,Glutamax I (L-alanyl-L-glutamine), Penicillin/streptomycin, Stem CellFactor (SCF) at 25 ng/ml, Thrombopoietin (TPO) (e.g., at 25 ng/ml),Fms-related tyrosine kinase 3 ligand (FL) (e.g., at 25 ng/ml),Interleukin-3 (IL-3) (e.g., at 10 ng/ml), Interleukin-6 (IL-6) (e.g., at10 ng/ml), and Heparin (e.g., at 5 Units/ml).

In another aspect, the disclosure provides methods of producingmegakaryocytes (such as those utilized in a method of producingplatelets or for another purpose) which may be generated by the steps of(a) culturing pluripotent stem cells to form hemogenic endothelial cells(PVE-HE); (b) culturing the hemogenic endothelial cells to form MLPs;and (c) culturing the MLPs to form megakaryocytes as shown in Examples 1and 2. Steps (a) and (b) may be carried out as described in thepreceding paragraph. Step (c) may be carried out in Iscove's ModifiedDulbecco's Medium (IMDM) as basal medium, human serum albumin,iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, TPO (e.g., at 30 ng/ml), SCF(e.g., at 1 ng/ml), IL-6 (e.g., at 7.5 ng/ml), IL-9 (e.g., at 13.5ng/ml), and optionally a ROCK inhibitor such as but not limited toY27632 (e.g., at 5 μM), and/or Heparin (e.g., at 5-25 units/ml).

In another aspect, the disclosure provides a pharmaceutical preparationcomprising platelets produced by the methods above. The preparation maybe suitable for use in a human patient and/or may comprise at least 10⁸platelets, and/or may be substantially free of leukocytes.

In another aspect the disclosure provides the use of the platelets ofany composition described herein or produced by any method hereindescribed in the manufacture of a medicament for the treatment of apatient in need thereof or suffering from a disease or disorderaffecting clotting.

In another aspect the disclosure provides a method of treating a patientin need of platelet transfusion, comprising administering platelets ofany composition described herein or produced by any method hereindescribed to said patient, which may be in an amount effective to treata disease or disorder affecting clotting and/or other platelet functionand/or another disorder that may be treated thereby, such asthrombocytopenia or trauma. Thrombocytopenia results from disturbancesin platelet production, distribution, or destruction. It is frequentlyfound in a variety of medical conditions, including liver cirrhosis, HIVinfection, autoimmune disease, idiopathic thrombocytopenia purpura,chemotherapy-induced myelosuppression and bone marrow disorders. A lowplatelet count is associated with an increased risk of bleeding. Anadditional exemplary disease or disorder that may be treated thereby ismalaria and other parasitic infections, which while not intending to belimited by theory is thought to be mediated by the ability of the humanplatelet factor 4 to kill malaria parasites within erythrocytes byselectively lysing the parasite's digestive vacuole (see Love et. al.,Cell Host Microbe 12 (6): 815-23, which is hereby incorporated byreference in its entirety).

In another aspect the disclosure provides a method of drug delivery,comprising administering platelets of any composition described hereinor produced by any method herein described to said patient, wherein saidplatelets deliver said drug. For example, it is thought that due totheir in vivo life span, lack of engraftment post administration, andhoming properties, platelets may be useful as a drug carrier. hESCs,hiPCs and MLPs may be genetically modified and used to produce plateletsthat express a desired drug for treatment of a disease. In one aspect,hESCs, hiPCs or MLPs could be genetically modified to express anantitumor agent. Platelets produced from such genetically modifiedhESCs, hiPSCs and MLPs may be used to deliver such antitumor agent to atumor for the treatment of a neoplastic disease.

The platelets of the present invention can be engineered to include oneor more therapeutic agents which are released by the platelets either ina passive manner (diffuse out of the platelet over time) or in an activemanner (are released upon activation and degranulation of platelets). Awide range of drugs can be used. The engineered platelets may beprepared so that they include one or more compounds selected from thegroup consisting of drugs that act at synaptic and neuroeffectorjunctional sites; drugs that act on the central nervous system; drugsthat modulate inflammatory responses; drugs that affect renal and/orcardiovascular function; drugs that affect gastrointestinal function;antibiotics; anti-cancer agents; immunomodulatory agents; drugs actingon the blood and/or the blood-forming organs; hormones; hormoneantagonists; agents affecting calcification and bone turnover, vitamins,gene therapy agents; or other agents such as targeting agents, etc.

In certain embodiments, the platelets have been engineered to includeone or more therapeutic agents, such as a small molecule drug, aptameror other nucleic acid agent, or recombinant proteins, e.g., which may bestored in the platelets' granules (α-granules, for example), andpreferably released upon activation of the platelets.

In certain embodiments, the platelets include one or more exogenousagents which promote or accelerate normal wound healing, reducescarring, reduce fibrosis, or a combination thereof.

In certain embodiments, the platelets include one or more exogenousanti-fibrotic agents. Platelets engineered to deliverantithrombotic/antirestenosis agents can be used use during angioplastyand thrombolysis procedures. In certain embodiments, the engineeredplatelets can used to prevent or reduce the severity of atherosclerosis.In certain embodiments, the engineered platelets can used to prevent orreduce the severity of restenosis. In still other embodiments, theengineered platelets can used as part of a treatment for solid tumors.The engineered platelets may include one or more immunostimulatoryagents.

A further aspect of the disclosure provides a method of producing areduced immunogenicity or “universal” platelet. In an exemplaryembodiment, the invention provides a β2 microglobulin-deficient plateletwhich can be generated using a pluripotent cell engineered to bedeficient in β2 microglobulin expression, such as a β2 microglobulinknockout pluripotent cell. The disclosure also provides a β2microglobulin-deficient platelet, megakaryocyte, PVE-HE or plateletprogenitor lacking expression of β2 microglobulin. A β2microglobulin-deficient platelet generally has low or preferablyundetectable Class I MHC molecules present in its plasma membrane,thereby reducing immunogenicity of the platelet. In other embodiments,the stem cell, and the resulting platelets, MKs, MK progenitors, PVE-HEand the like, have been engineered to lack expression (or at least havereduced expression) of one or more of β2 microglobulin, HLA-A, HLA-B,HLA-C, TAP1, TAP2, Tapasin, CTIIA, RFX5, TRAC, or TRAB genes (orproteins, depending on the readout employed). There are a variety oftechniques for engineering cells to eliminate or reduce the expressionof one or more genes (or proteins), including the use of zinc-fingernucleases (ZFNs), transcription activator-like effector nucleases(TALENs), and CRISPR/Cas-based methods for genome engineering, as wellas the use of transcriptional and translational inhibitors such asantisense and RNA interference (which can be achieved using stablyintegrated vectors and episomal vectors).

In another aspect, a method is provided for producing platelets frommegakaryocytes or MLPs comprising culturing a non-adherent population ofmegakaryocytes of MLPs under shear force conditions in the presence of aprotease inhibitor, and harvesting and optionally isolating plateletsfrom the culture.

The protease inhibitor may be an MMP inhibitor.

The shear force conditions may be constant shear force conditions. Theshear force conditions may comprise a shear force of 1-4.1 dynes/cm².

The megakaryocytes or MLPs may be cultured in a microfluidic device.

The megakaryocytes or MLPs may be derived from iPS cells, ES cells, ornaturally occurring CD34⁺ cells, optionally bone marrow or umbilicalcord blood CD34⁺ cells.

The protease inhibitor may be an MMP inhibitor such as GM6001.

The protease inhibitor may be an MMP8 specific inhibitor such as MMP8-I((3R)-(+)-[2-(4-Methoxybenzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-hydroxamate]).

The protease inhibitor may be two or more protease inhibitors.

The two protease inhibitors may be an MMP general (pan) inhibitor and anMMP8 specific inhibitor.

The protease inhibitor may be added at a time of peak production ofplatelets within the culture.

The megakaryocytes or MPLs may be cultured in the presence of TPO or aTPO agonist to cause the formation of proplatelets, wherein theproplatelets release platelets. The megakaryocytes or MPLs are culturedin hematopoietic expansion medium and optionally in (1) TPO or a TPOagonist, SCF, IL-6 and IL-9 or (2) TPO or a TPO agonist, SCF, and IL-11to cause the formation of pro-platelets in culture, wherein thepro-platelets release platelets.

The megakaryocytes or MPLs may be cultured at a temperature greater than37° C. and equal to or less than 40° C.

The megakaryocytes or MPLs may be cultured at a temperature of about 39°C.

In another aspect, a method is provided for producing platelets frommegakaryocytes or MPLs comprising culturing a non-adherent population ofmegakaryocytes or MPLs derived from iPS cells or ES cells at atemperature greater than 37° C. and equal to or less than 40° C., andharvesting and optionally isolating platelets from the culture.

The megakaryocytes or MPLs may be cultured at a temperature of about 39°C.

In another aspect, a method is provided for producing MPLs from PVE-HEcells comprising culturing a population of PVE-HE cells derived from iPScells or ES cells in the presence of an inhibitor of BET, and harvestingand optionally isolating MPLs from the culture.

The inhibitor of BET may be I-BET151.

The inhibitor of BET may be added to the PVE-HE cells in the last 48hours, last 36 hours, last 24 hours, last 18 hours, last 12 hours, orlast 6 hours of culture.

In another aspect, a method is provided for producing MPLs from PVE-HEcells comprising culturing a population of PVE-HE cells derived from iPScells or ES cells in the presence of a c-myc suppressor or inhibitor,and harvesting and optionally isolating MPLs from the culture.

The c-myc suppressor or inhibitor may be added to the PVE-HE cells inthe last 48 hours, last 36 hours, last 24 hours, last 18 hours, last 12hours, or last 6 hours of culture.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Stepwise process depicting the generation of platelets frompluripotent stem cells. This Figure shows the progression ofdifferentiation from pluripotent stem cells through pluripotent-derivedhemogenic endothelial cells (PVE-HE) through megakaryocytelineage-specific progenitor cells (MLP) and through megakaryocytes (MK).

FIG. 2A-C. Progression of differentiation of iPS cells through advanceddifferentiation morphology cells (ADM). This Figure shows iPS cellprogression toward PVE-HE. FIG. 2A, after 48 hours attached cells showtypical pluripotent stem cell morphology under feeder-free condition.FIG. 2B, the almost complete transition from pluripotent stem cellmorphology into scattered small cell clusters is shown. FIG. 2C, after96 to 146 hours post PVE-HE differentiation initiation, advanceddifferentiation morphology was observed showing small compact cellclusters growing on top of the monolayer.

FIG. 3A-B. Characterization of advanced differentiation morphology ofPVE-HE. This Figure shows the phenotype and morphology of ADM cells thathave differentiated into PVE-HE. The ADM cells show the PVE-HE phenotypeCD31⁺CD144(VE-Cad)⁺CD105⁺ at this stage of differentiation (FIG. 3A).ADM cells were also analyzed for morphologic changes (FIG. 3B).

FIG. 4. Characterization of iPS-PVE-HE-derived megakaryocyticlineage-specific progenitors (MLPs) by flow cytometry. This Figure showsthe phenotype of human iPS-PVE-HE-MLP cells asCD34⁺CD31⁺CD41a⁺CD43⁺CD13⁺CD14⁻CD42b^(−/+).

FIG. 5A-C. Morphologic analysis of human iPS-PVE-HE-MLP-derived maturingMK cells. This Figure shows nuclei inside MK cells (indicated by “N”),and readily observable proplatelet forming cells with elongatedpseudopodia (indicated by arrows). At 72 hours post initiation ofplatelet differentiation, very large polyploidy MKs (50 μM) becameabundant with progression of maturation (FIGS. 5A & 5B. Scale bar in 5Ais 100 μM, N in 5B indicates nucleus inside MK). From between 72-96hours proplatelet forming cells with elongated pseudopodia were readilyobserved microscopically. (FIGS. 5A & 5C, indicated by arrows).

FIG. 6A-E. Comparison of phenotype and purity of platelet preparationsfrom different sources. This Figure shows flow cytometric analysis ofthe morphology and cell surface expression of CD41a and CD42b onperipheral blood derived human platelets and iPS-PVE-HE-MLP-MK-derivedplatelets. Between 72-96 hours post initiation, the amount ofCD41a+CD42b+platelets increased dramatically, reaching levels as high asabout 70% (FIG. 6D). FIGS. 6A-6B show forward scatter (“FSC-A”) and sidescatter (“SSC-A”) for donor-derived human platelets (6A), andhES-derived platelets (hES-PVE-HE-MLP) produced as described in Example3 (6B). FIGS. 6C-E show expression of CD41a and CD42B by donor-derivedhuman platelets (6C), iPS-derived platelets (iPS-PVE-HE-MLP) (6D), andhES-derived platelets (hES-PVE-HE-MLP) (6E), with the latter two sampleshaving been produced as described in Example 3.

FIG. 7. Ultrastructural comparison of peripheral blood derived humanplatelets and platelets produced by differentiation of human inducedpluripotent cells (hiPSC-PLTs) by transmission scanning microscopy. ThisFigure shows the similarities in cellular characteristics of peripheralblood derived human platelets and hiPSC-PLTs of the present disclosure.The hiPSC-PLTs are discoid.

FIG. 8. Comparison of peripheral blood derived human platelets (hPRP)and hiPSC-PLTs. This Figure shows similarities between the two cellpreparations in platelet diameter (left panel), and expression ofstructural cell proteins beta1-tubulin and F-actin (via FITC-phalloidinbinding) (right panel), which are involved in activation inducedplatelet shape changes. Negative Hoechst staining (top right) confirmedthe absence of nuclear DNA in iPSC-PLTs and donor-derived PLTs.

FIG. 9. Comparison of peripheral blood derived human platelets andhiPSC-PLTs. This Figure shows the similarities in morphologiccharacteristics of peripheral blood derived human platelets andhiPSC-PLTs of the present disclosure using differential interferencecontrast (DIC) live-cell microscopy. Both the peripheral blood derivedhuman platelets and the hiPSC-PLT show pseudopodia emission indicativeof activation when bound to the negatively charged glass surface.

FIG. 10A-B. Comparison of peripheral blood derived human platelets andhiPSC-PLTs. This Figure shows the similarities of alpha-granuleexpression as demonstrated by Thrombospondin 4 (TSP4) and PlateletFactor 4 (PF4) labeling. TSP4 and PF4 are chemokines released fromalpha-granules of activated platelets. hiPSC-PLTs (FIG. 10B) were seento have normal alpha-granule expression relative to normal humanplatelets (FIG. 10A), as demonstrated by TSP4 and PF4 labeling.

FIG. 11. Functional evaluation of hiPSC-PLTs. This Figure shows the invitro activation of hiPSC-PLTs with thrombin as measured by theupregulation of two adhesion molecules CD62p and αIIbβIII (as measuredby the PAC-1 Ab).

FIG. 12A-B. Functional comparison of circulating human platelets andplatelets derived from human iPSCs and ESCs. This Figure shows the invivo clot forming potential of circulating human platelets, hiPSC-PLTsand hESC-PLTs. Experiments were performed with natural human platelets(“hPLT”), iPSC-PLT, and hES cell derived platelets (“hESC-PLT”). FIG.12A shows representative images of thrombi formed in a mouse vessel wallinjury model. FIG. 12B graphically illustrates the average number ofplatelets bound to the thrombus in each experiment. Platelet binding wasinhibited by treatment with ReoPro, an anti-αIIbβIII antibody fragment,indicating that binding was dependent on αIIbβIII as expected. (FIG.12B).

FIG. 13. Kinetics of PLTs in macrophage-depleted NOD/SCID mice afterinfusion. This Figure shows a detectable circulation of hiPSC-PLTs andhESC-PLTs of eight hours.

FIG. 14A-E. Exemplary process flow diagram for production of plateletsfrom pluripotent stem cells.

FIG. 15. FACS Analysis, CD41a, CD42b—MLP Derived From hiPSC.

FIG. 16. Representative MLPs Derived from hiPSC.

FIG. 17. FACS Analysis CD31+, CD43+—MLP Derived from hiPSC.

FIG. 18. Representative MKs derived from hESC.

FIG. 19. Proplatelet Formation from MK.

FIG. 20. FACS Analysis CD41a, CD42b.

FIG. 21. DIC Microscopy with β1-Tubulin Staining, Human Donor PLT (top)hESC-PLT (bottom).

FIG. 22. Platelet purity as a function of time in the presence of DMSO(diamonds), MMP inhibitor GM6001 (squares), and GM6001 and 8% dextran(referred to as “viscosity”) (triangles), under constant shear forceculture conditions. The x-axis corresponds to sample number, with asample removed every 30 minutes over a 6+ hour culture.

FIG. 23. Platelet numbers as a function of time in the presence of DMSO(diamonds), MMP inhibitor GM6001 (squares), and GM6001 and 8% dextran(referred to as “viscosity”) (triangles), under constant shear forceculture conditions. GM6001 was added to the culture at day 0.

FIG. 24. Platelet numbers in the presence of DMSO (left bar) and MMPinhibitor GM6001 (middle bar) under constant shear force cultureconditions, and under static culture conditions (in the absence of MMPinhibitor GM6001) (right bar).

FIG. 25. Platelet purity as a function of time in the presence of MMPinhibitor GM6001 in a microfluidic device at a flow rate of 12microliters/min (squares) and at a flow rate of 16 microliters/min(triangles). The control (diamonds) is in the presence of DMSO since theMMP inhibitor is dissolved in DMSO.

FIG. 26. Platelet numbers as a function of flow rate. Left bar: 12microliters/min, Right bar: 16 microliters/min.

FIG. 27. Platelet purity as a result of static culture in the presenceof MMP8 specific inhibitor MMP8-I (second bar), pan-MMP inhibitor GM6001(third bar), or the combination of MMP8-I and GM6001 (fourth bar). TheMMP8-I is(3R)-(+)-[2-(4-Methoxybenzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-hydroxamate],and it is commercially available from Millipore. The first bar is thecontrol.

FIG. 28. Platelets numbers as a result of static culture in the presenceof MMP8 specific inhibitor MMP8-I (second bar), pan MMP inhibitor GM6001(third bar), or the combination of MMP8-I and GM6001 (fourth bar). Thefirst bar is the control.

FIG. 29. Platelet purity as a function of time in culture at 37° C.(left bar per pair) and 39° C. (right bar per pair). The temperature wasset at the beginning of and maintained throughout the culture.

FIG. 30. Platelet number as a function of time in culture at 37° C.(left bar per pair) and 39° C. (right bar per pair).

FIG. 31. Megakaryocyte progenitor (MLP) numbers harvested at day 6+4(i.e., day 10 of differentiation) as a function of increasing dose ofiBET-151 (in μM). iBET is added to the culture at the 6+3 day timeframe, and MLPs are exposed to iBET for a period of about 24 hours priorto their harvest. iBET concentrations: 0 (left bar), 0.1 microM (middlebar), 0.25 microM (right bar).

FIG. 32. Relative quantitative analysis of mRNA of c-myc and GATA-1 onday 6+4 as a function of increasing dose of iBET-151. iBETconcentrations: 0 (left bar), 0.1 microM (middle bar), 0.25 microM(right bar) for each triplet.

FIG. 33. CD14+ cell purity in cell population harvested at day 6+4 as afunction of increasing dose of iBET-151. iBET concentrations: 0 (leftbar), 0.1 microM (middle bar), 0.25 microM (right bar).

DETAILED DESCRIPTION OF INVENTION

Lists of definitions and abbreviations used in this disclosure areprovided at the end of the Detailed Description.

As noted above, limitations in the supply of platelets can havepotentially life-threatening consequences for transfusion-dependentpatients. Pluripotent cells can be propagated in vitro indefinitely, andrepresent a potentially inexhaustible and donorless source of plateletsfor human therapy. The ability to create banks of hESC lines withmatched or reduced incompatibility could potentially decrease oreliminate the need for immunosuppressive drugs and/or immunomodulatoryprotocols. Exemplary embodiments provide a method comprising producingpatient-specific iPS cells (for example using methods herein describedor any others known in the art), and producing patient-specificplatelets from said patient-specific iPS cells, which platelets may beused for treatment of patients, such as patients who have developed orare at risk of developing platelet alloimmunity.

Exemplary embodiments provide an efficient method to generatemegakaryocytes (MKs) from pluripotent stem cells under serum-free andfeeder-free conditions. Preferably the MKs are produced frommegakaryocyte lineage-specific progenitor cells (MLPs, which are furtherdescribed herein). Preferably the MKs are not produced fromhemangioblasts or hemangio-colony forming cells such as those disclosedin U.S. Pat. No. 8,017,393. Using methods disclosed herein, pluripotentstem cells (iPSC and ESCs) were directed towards MK differentiation. Theefficiency of differentiation of megakaryocytes from MLPs has been veryhigh (up to 90%). Without further purification, 85% of live cells fromthe MK suspension cultures were CD41a+ CD42b+ and the mature MKs werealso CD29+ and CD61+. These in vitro-derived MK cells can undergoendomitosis and become mature, polyploid MKs. Importantly, proplateletforming cells with elongated pseudopodia were observed at the late stageof MK culture, indicating that MKs generated in this system are able toundergo terminal differentiation and generate functional platelets underfeeder-free conditions.

Described herein is an efficient system which is adaptable for largescale and efficient in vitro megakaryocyte production using iPSC orother pluripotent stem cells as source cells under controlledconditions. Additionally, the platelets may be produce in sufficientquantities to supplement or supplant the need for donor-derivedplatelets. Additionally, the disclosed methods may be utilized toproduce platelets and platelet progenitor cells in a predictable manner,such that the cells may be produced “on demand” or in quantities desiredto meet anticipated need. The cells expressed CD41a and CD42b, underwentendomitosis, and formed mature polyploid MKs. Upon further maturation,they generated functional or activated platelets that were stimulated bythrombin to become positive for cell adhesion molecules, CD62p andαIIbβIII (thought to occur by exposure of the PAC-1 binding site afterconformational change of αIIbβIII upon activation, while CD62p isthought to be exposed on outside membrane due to granule release). Bothof these markers are known to be expressed on the surface of activatedplatelets and were detected on the hiPSC-PLTs using a PAC-1 and CD62p(p-selectin) binding assay. Because no stromal inducer cells are neededfor megakaryocyte production, the methods described herein can providefor feeder-free platelet generation, such as platelet generation withoutany use of xenogeneic cells.

In exemplary embodiments, additional factors including estradiol,vitamin B3, matrix metalloproteinase inhibitors (MMP), inhibitors ofc-myc expression, and extracellular matrix proteins may also be used toenhance platelet production, such as by stimulating megakaryocytematuration and/or stimulating platelet production, which may be carriedout in the absence of stromal cells.

The production of megakaryocytes under serum-free and stromal freeconditions may allow screening for factors that are critical inregulating megakaryopoiesis and thrombopoiesis under well-definedconditions. Factors so identified may contribute to clinicalapplications. Advances in this area may also likely provide insightsinto the cellular and molecular mechanisms regulating different aspectsof megakaryopoiesis including lineage commitment, expansion andmaturation.

Exemplary embodiments integrate step-wise inductions of megakaryocytedifferentiation from pluripotent stem cells. Further optimization andestablishment of in-process controls can be performed to improve theconsistency and efficiency of this system for clinical applications. Theunderlying cellular or extra-cellular mechanisms regulatingmegakaryocyte maturation need not be completely defined in order topractice these methods. Other factors that promote polyploidization andcytoplasmic maturation may be identified and included to facilitate theterminal differentiation of in vitro-derived megakaryocytes. Forinstance, at least one ROCK kinase inhibitor may be used to induceendomitosis of megakaryocytes at an early stage. However, this effect isthought to be likely due to an artificial blocking of chromosomesegregation and cytokinesis rather than an orchestrated cellular andnuclear maturation of a differentiating megakaryocyte. It may beadvantageous to reach a balance between the expansion, endomitosis andthe cytoplasmic maturation to achieve the highest in vitro megakaryocyteyields, terminal differentiation status and downstream production offunctional platelets under defined conditions.

These current results demonstrated that platelets derived frompluripotent stem cells share morphological and functional properties ofnormal blood platelets. These pluripotent stem cell derived humanplatelets are able to function in vivo as well.

Additional hemodynamic events may occur during the generation andpropagation of platelet thrombi in the living organism which may not befully mimicked by in vitro systems. The availability of intravitalimaging technology provides a means to directly examine and quantify theplatelet-dependent thrombotic process that occurs after vascular injuryin complex in vivo systems. Using intravital high-speed widefieldmicroscopy, the inventors demonstrated that pluripotent stemcell-derived platelets are incorporated into the developing mouseplatelet thrombus at the site of laser-induced arteriolar wall injury inliving mice similarly to normal human blood platelets. Pretreatment ofthe pluripotent cell-derived and control platelets with ReoPro markedlyreduced the number of both donated and pluripotent stem cell-derivedplatelets incorporating in the thrombi, confirming the binding wasmediated by αIIbβIII integrin. These results indicate that pluripotentstem cell-derived platelets are functional at the site of vascularinjury in living animals.

Platelets are anucleate cells that adhere to tissue and to each other inresponse to vascular injury. This process is primarily mediated by theplatelet integrin αIIbβIII, which binds to several adhesive substratessuch as von Willebrand Factor (vWF) and fibrinogen to bridge and furtheractivate platelets in a growing thrombus (36). The results hereindemonstrate that platelets generated from pluripotent stem cells arefunctionally similar to normal blood platelets both in vitro and inliving animals. The pluripotent stem cell-derived platelets were shownto possess important functions involved in hemostasis, including theability to aggregate when stimulated with physiological agonists. Inaddition, immunofluorescence and transmission electron microscopicresults further demonstrate that platelets generated from pluripotentstem cells are similar to normal blood platelets.

As further described in the examples below, numerous similaritiesbetween pluripotent cell-derived platelets and purified normal humanplatelets were observed. These similarities include the following.

hiPSC-PLTs are discoid (as demonstrated by transmission electronmicroscopy)

hiPSC-PLTs are mostly ultrastructurally identical to circulating humanPLTs (as demonstrated by transmission electron microscopy).

The size of hiPSC-PLTs is comparable to that of circulating human PLTs(2.38 μm±0.85 μm versus 2.27 μm±0.49 μm) as demonstrated by DIC andβ1-tubulin IF microscopy)

hiPSC-PLTs were able to spread on glass and form both filopodia andlamelopodia as demonstrated by DIC live-cell microscopy images).

hiPSC-PLTs are anucleate—comparable to circulating human PLTs (asdemonstrated by Hoechst labeling).

hiPSC-PLTs have a normal tubulin cytoskeleton relative to circulatinghuman PLTs (as demonstrated by β1-tubulin labeling).

hiPSC-PLTs have normal filamentous actin relative to circulating humanPLTs (as demonstrated by phalloidin labeling).

hiPSC-PLTs have normal alpha-granule expression relative to circulatinghuman PLTs (as demonstrated by TSP4 and PF4 labeling).

Another scientific and clinical issue is whether pluripotent stemcell-derived platelets are functional in the complex in vivo setting. Alarge number of experimental models were established in the past decadeto investigate thrombus formation in mice, including the laser-injurythrombosis model recently used by several groups (37:38:39). Thelaser-induced thrombosis model initiates platelet thrombus formation asfast as 5-30 seconds following injury. Therefore, this model allows themonitoring of the real-time incorporation of rapidly cleared humanplatelets and hESC-PLTs into the developing mouse platelet thrombus,which involves a large number of signaling pathways, enzymatic cascades,as well as the interplay of a myriad of cellular and protein components.This model also mirrors the inflammatory reactions associated withthrombin-induced thrombosis.

Using the laser-induced vessel injury model, the intravital microscopyanalyses demonstrate that hiPSC-PLTs and hESC-PLTs, like bloodplatelets, are incorporated into the developing mouse platelet thrombusthrough αIIbβIII integrin following vascular injury (FIG. 12A).hiPSC-PLTs and hESC-PLTs functional capabilities were determined to bemediated by αIIbβIII by pretreatment with ReoPro, a Fab fragment of ahuman-murine chimeric monoclonal antibody that binds specifically toαIIbβIII and inhibits platelet function (FIG. 12B). These resultsprovide evidence that hESC-PLTs are functional at the site of vascularinjury in vivo. Importantly, it is shown for the first time thatplatelets derived from pluripotent stem cells under serum and feederfree conditions are able to facilitate coagulation and thrombusformation in vivo.

Two previous studies have reported the generation of MKs from hESCs. Theyield in these systems is extremely low, and unlike the current system,relies on co-culture with animal stromal cells supplemented with serum(15,16). Moreover, in vivo functionality was not reported (15,16). Theelimination of these two variables in pluripotent stem celldifferentiation allows the generation of platelets without exposure toanimal products. Additionally, the present disclosure demonstrates thatthe feeder-free system described herein can generate MKs with highefficiency and that functional platelets can be efficiently generatedunder the feeder-free conditions.

Thrombopoiesis is a highly complex process, with sophisticatedreorganization of membrane and microtubules and precise distributions ofgranules and organelles (40). Despite recent advances in theunderstanding of platelet biogenesis, mechanistic details underlyingmembrane reorganization, initiation of proplatelets, transportation ofplatelet organelles and secretary granules, and control of platelet sizeremain to be elucidated. The ability to generate MKs under serum- andfeeder-free conditions should aid in the screening of factors that arecritical in regulating different aspects of megakaryopoiesis underwell-defined conditions, including lineage commitment, expansion andmaturation.

This disclosure provides various methods for producing PVE-HE cells,MLPs, MK, proplatelets and platelets in vitro (or ex vivo) that areiPS-derived or ESC-derived.

This disclosure provides methods for transitioning from an iPS cell orES cell to a PVE-HE cell, or to a MLP, or to a MK, or to a platelet.This disclosure provides methods for transitioning from a PVE-HE cell toa MLP, or to a MK, or to a platelet. This disclosure provides methodsfor transitioning from a MLP to a MK, or to a platelet. This disclosureprovides methods for transitioning from a MK to a platelet. Thesevarious cultures are described briefly below.

PVE-HE cells may be produced from iPS or ES cells through a method thatcomprises culturing iPS cells or ES cells in a culture medium comprisingIscove's Modified Dulbecco's Medium (IMDM) as basal medium, human serumalbumin, iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, and further comprising bonemorphogenetic protein 4 (BMP4) (e.g., at 50 ng/ml), basic fibroblastgrowth factor (bFGF) (e.g., at 50 ng/ml), and vascular endothelialgrowth factor (VEGF) (e.g., at 50 ng/ml). This culture period may lastan average of 6 days.

MLPs may be produced from PVE-HE cells through a method that comprisesculturing PVE-HE cells in a culture medium comprising Iscove's modifiedDulbecco's medium (IMDM), Ham's F-12 nutrient mixture, Albucult (rhAlbumin), Polyvinylalcohol (PVA), Linoleic acid, SyntheChol (syntheticcholesterol), Monothioglycerol (a-MTG), rhInsulin-transferrin-selenium-ethanolamine solution, protein-freehybridoma mixture II (PFHMII), ascorbic acid 2 phosphate, Glutamax I(L-alanyl-L-glutamine), Penicillin/streptomycin, and further comprisingStem Cell Factor (SCF) (e.g., at 25 ng/ml), Thrombopoietin (TPO) (e.g.,at 25 ng/ml), Fms-related tyrosine kinase 3 ligand (FL) (e.g., at 25ng/ml), Interleukin-3 (IL-3) (e.g., at 10 ng/ml), Interleukin-6 (IL-6)(e.g., at 10 ng/ml), and optionally Heparin (e.g., at 5 Units/ml). Thisculture period may last an average of 3-4 days. In the latter half ofthe culture, including in the last 48 hours, the last 36 hours, the last24 hours, the last 18 hours, the last 12 hours, or the last 6 hours aninhibitor of BET may be added to the culture, preferably atsub-cytotoxic levels. The MLPs harvested from these cultures may becryopreserved or used immediately for platelet production or otheranalysis.

Megakaryocytes may be produced from MLPs through a method that comprisesculturing the MLPs in a medium that comprises Iscove's ModifiedDulbecco's Medium (IMDM), human serum albumin, iron-saturatedtransferrin, insulin, b-mercaptoethanol, soluble low-density lipoprotein(LDL), cholesterol, and further comprises TPO (e.g., at 30 ng/ml), SCF(e.g., at 1 ng/ml), IL-6 (e.g., at 7.5 ng/ml), IL-9 (e.g., at 13.5ng/ml), and optionally a ROCK inhibitor such as Y27632 (e.g., at 5 μM),and/or Heparin (e.g., at 5-25 units/ml).

Megakaryocytes may be produced from MLPs through a method that comprisesculturing the MLPs in a medium that comprises Iscove's ModifiedDulbecco's Medium (IMDM), human serum albumin, iron-saturatedtransferrin, insulin, b-mercaptoethanol, soluble low-density lipoprotein(LDL), cholesterol, and further comprises one or more of TPO (e.g., at10-100 ng/ml), SCF (e.g., at 0.5-100 ng/ml), IL-11 (e.g., at 5-25ng/ml), and optionally a ROCK inhibitor such as Y27632 (e.g., at 5 μM),and/or Heparin (e.g., at 2.5-25 Units/ml).

This latter culture produces MK and platelets depending on the length ofthe culture. Platelets are typically observed by about days 3-4 ofculture. It will be understood that during the culture period the MLPwill be maturing into MK, and the MK will be maturing into proplatelets,and the proplatelets will be maturing into platelets.

Platelets therefore may be produced from MLPs or MKs through a methodthat comprises culturing the MLPs or MKs in a medium that comprisesIscove's Modified Dulbecco's Medium (IMDM), human serum albumin,iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, and further comprises TPO(e.g., at 30 ng/ml), SCF (e.g., at 1 ng/ml), IL-6 (e.g., at 7.5 ng/ml),IL-9 (e.g., at 13.5 ng/ml), and optionally a ROCK inhibitor such asY27632 (e.g., at 5 μM), and/or Heparin (e.g., at 5-25 units/ml). Theseculture period may be 4-8 days in length, or more.

Platelets may be produced from MLPs or MKs through a method thatcomprises culturing the MLPs or MKs in a medium that comprises Iscove'sModified Dulbecco's Medium (IMDM), human serum albumin, iron-saturatedtransferrin, insulin, b-mercaptoethanol, soluble low-density lipoprotein(LDL), cholesterol, and further comprises one or more of TPO (e.g., at10-100 ng/ml), SCF (e.g., at 0.5-100 ng/ml), IL-11 (e.g., at 5-25ng/ml), and optionally a ROCK inhibitor such as Y27632 (e.g., at 5 μM),and/or Heparin (e.g., at 2.5-25 Units/ml).

These platelet production methods include culture of MLP or MK understatic or shear force culture conditions. A static culture condition isa culture condition in which the culture medium in contact with thecultured cells is relatively static. A shear force culture condition isa culture condition that comprises deliberate and constant movement ofculture medium in contact with the cultured cells. Shear force ismeasured in dynes/cm². In some instances, the shear force approximatesthe shear force that occurs in the hematopoietic environment of the bonemarrow. The shear force in the BM sinusoid has been reported to be about1.3 to 4.1 dynes/cm². The disclosure contemplates that a shear forceculture may be carried out at a shear force ranging from about 1 toabout 4.5 dynes/cm², or about 1.3 to about 4.1 dynes/cm², including atany force or any range of forces therebetween including about 1.5, about2.0, about 2.5, about 3.0, about 3.5, about 4.0, and about 4.1dynes/cm².

It will be understood that the culture may be carried out at a flow ratethat yields such shear forces. For a given culture, the shear force isassociated with flow rate (volume/time). Shear force can be determinedwith knowledge of the flow rate, for any given culture or culturedevice, using knowledge in the art. Some of the experimental resultsprovided herein compare platelet production in a given microfluidicdevice at flow rates of 12 microliters/min and 16 microliters/min. Insome instances, the flow rate may be in the range of 5-25microliters/min, or within 10-20 microliters/min. In some instances, theflow rate may be in the range of 10-15 microliters/min and in others itmay be in the range of 16-20 microliters/min.

Where a shear force culture is used, the platelet production method maycomprise a first culture period in a static culture for 3-4 days, oruntil platelets are observed, followed by a second culture in a shearforce environment such as a microfluidic device or other device capableof inducing a shear force.

It is to be understood that aliquots may be harvested from such culturesand measured for platelet content using for example FACS. This willidentify periods of peak platelet production.

It is also be understood that platelet production methods often timeshave as a starting material a mixture of MLPs and MK, or comprise intheir culture medium at some point during the culture period a mixtureof MLPs and MK.

The disclosure contemplates that the MLP or MK cultured in microfluidicdevices or other culture devices in which a shear force condition can beachieved. In some instances, the device is designed such that the MLP orMK are immobilized within but not attached to the device. In someinstances, the device is made of a synthetic resin such aspolydimethylsiloxane (PDMS) or dimethicone. These are commonly used inmicrofluidic devices and chips.

The disclosure further contemplates that, when platelets are producedusing shear force culture conditions, better platelet yields andfunctionality is obtained if particular protease inhibitors are added tothe culture. It was found according to this disclosure that whenplatelets are placed under shear force cell surface CD42b can be lostfrom the platelet surface rendering the platelet less active. To avoidthis and yet still reap the benefits of a shear culture, the disclosurecontemplates using a protease inhibitor that prevents the shearing orloss of CD42b. Examples of such inhibitors include metalloproteaseinhibitors and more specifically matrix metalloprotease (MMP)inhibitors. Another example of inhibitors that may be used in the shearculture is plasminogen activator inhibitors. These inhibitors may be paninhibitors, intending that a single inhibitor may inhibit more than oneand possibly all proteases within the class. Alternatively, they may bespecific inhibitors, intending that a single inhibitor inhibits oneprotease within the class altogether or predominantly.

In some instances, the cultures are performed in the presence of an MMPinhibitor. The inhibitors may be small molecules such as small organicmolecules, antibodies or antibody fragments, antisense or RNAi nucleicacids, and the like.

Examples of MMP inhibitors include, but are not limited to, GM6001 (apan inhibitor), N-Dansyl-D-phenylalanine, 4-epi-Chlortetracycline,Hydrochloride Pyridoxatin, ARP 100, ARP 101, Batimastat, Chlorhexidine,Dihydrochloride, cis-ACCP, CL 82198 hydrochloride, Minocycline,Hydrochloride, Alendronate, Sodium Salt, GM 1489, TAPI-1, TAPI-2, GM6001, Marimastat, MMP Inhibitor II, MMP Inhibitor III, EGTA, MMPInhibitor V, MMP-13 Inhibitor, MMP-2 Inhibitor I, MMP-2 Inhibitor II, CP471474, MMP-2/MMP-3 Inhibitor I, MMP-2/MMP-3 Inhibitor II, MMP-2/MMP-9Inhibitor I, MMP-2/MMP-9 Inhibitor II, MMP-2/MMP-9 Inhibitor V. Ecotin,E. coli, MMP-3 Inhibitor, MMP-3 Inhibitor III, MMP-3 Inhibitor IV,Actinonin, MMP-3 Inhibitor V, MMP-3 Inhibitor VIII, MMP-7 AntisenseOligonucleotide, Sodium Salt, MMP-8 Inhibitor I, MMP-9 Inhibitor I,MMP-9/MMP-13 Inhibitor I, MMP-9/MMP-13 Inhibitor II, NNGH, NSC 23766,PD166793, Pro-Leu-Gly hydroxamate hydrochloride, Ro 32-3555, PF-356231,SB-3CT, Phosphoramidon, WAY 170523, UK 370106, UK 356618, Bariumchloride dehydrate, Luteolin, Isobavachalcone, Doxycycline Hyclate,Collagenase Inhibitor I, o-Phenanthroline, and TAPI-0 from Santa CruzBiotechnology, Inc. They also include TIMP-1, TIMP-2, TIMP-3, TIMP-4,GM6001, methylprednisolone, batimastat, marimastat, prinomastat, BAY12-9566, MMI270(B), BMS-275291, metastat and other inhibitors of MMP-1through MMP-26. It is to be understood that a MMP inhibitor may inhibitonly one MMP family member or it may inhibit more than one or all MMPfamily members.

Certain synthetic MMP inhibitors generally contain a chelating groupthat binds the catalytic zinc atom at the MMP active site tightly.Common chelating groups include hydroxamates, carboxylates, thiols, andphosphinyls.

Other MMP inhibitors include BB-94, Ro 32-3555, BB-1101, BB-2516, SE205,CT1746, CGS 27023A, AG3340, BAY 12-9566, D2163, D1927, PNU-142372,CMT-1, and actinonin.

Many MMP inhibitors are commercially available.

The art is familiar with MMP inhibitors, and further examples areprovided in U.S. Pat. Nos. 4,877,805; 5,837,224; 6,365,630; 6,630,516;6,683,069; 6,919,072; 6,942,870; 7,094,752; 7,029,713; 6,942,870;6,919,072; 6,906,036; 6,890,937; 6,884,425; 6,858,598; 6,759,432;6,750,233; 6,750,228; 6,713,074; 6,699,486; 6,645,477; 6,630,516;6,548,667; 6,541,489; 6,379,667; 6,365,630; 6,130,254; 6,093,398;5,962,466; 5,837,224; 7,705,164; 7,786,316; 8,008,510; 7,579,486;8,318,945; and 7,176,217; published U.S. patent applications20070037253; 20060293345; 20060173183; 20060084688; 20050058709;20050020607; 20050004177; 20040235818; 20040185127; 20040176393;20040067883; 20040048852; 20040034098; 20040034086; 20040034085;20040023969; 20040019055; 20040019054; 20040019053; 20040006137;20040006077; 20030212048; 20030004165; 20020198176; 20020177588;20020169314; 20020164319; 20020106339; 20020061866; 20020054922;20020049237; 20020010162; 20010039287; and 20010014688; and publishedPCT applications WO 02/064552, WO 05/1103399, WO 06/028523,WO2008/024784, WO 02/064552, WO 05/110399, WO 06/028523, WO01/62261, andWO2008/024784, each of which is incorporated herein by reference.

MMP inhibitors have also been described in the scientific literature,see, for example, Whittaker et al. Chem Rev. 99:2735-2776, 1999;Whittake et al. Celltransmissions 17(1):3-14 (Table AB) and Harrison,Nature Reviews Drug Discovery 6:426-427, 2007 (Table AC), the specificteachings of which are incorporated herein by reference.

Some MMP inhibitors may be

In some instances, an MMP8 inhibitor such as specific MMP8 inhibitor maybe used in a static or a shear force culture. In some instances, theMMP8 inhibitor may be used in cultures of naturally occurring MLP andMK, such as bone marrow or umbilical cord blood derived MLP (e.g., CD34⁺progenitor cells) or MK.

An example of an MMP8 specific inhibitor is MMP8-I having chemical name(3R)-(+)-[2-(4-Methoxybenzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-hydroxamate],and which is commercially available from Millipore.

In some instances, the protease inhibitor may be a plasminogen activatorinhibitor. Examples of plasminogen activator inhibitors include, but arenot limited to, plasminogen activator inhibitor 1 (PAI-1), plasminogenactivator inhibitor 2 (PAI-2), and tissue plasminogen activator (tPA)inhibitor. Other plasminogen activator inhibitors include thosedescribed in U.S. Pat. No. 4,923,807; international PCT applicationsWO/13063331; WO/1316974; and literature references Fortenberry Y M.Plasminogen activator inhibitor-1 inhibitors: a patent review(2006-present). Expert Opin Ther Pat. 2013 July; 23(7):801-15; andPannekoek et al., EMBO J. 1986; 5(10):2539-44 each of which isincorporated herein by reference.

In some instances, two or more protease inhibitors may be used togetherin a culture. As an example, the MMP inhibitor GM6001 may be usedtogether with the MMP8 specific inhibitor MMP8-I.

In some instances, the cultures may be performed at a temperature above37° C. The culture temperature may be in the range of 37° C. to 45° C.,or 37° C. to 42° C. or 38° C. to 41° C., or 39° C. to 40° C., or about39° C. or about 40° C. The culture is performed at the set temperature.Culture at a temperature above 37° C. is referred to herein as anelevated temperature culture.

In some instances, the method that produces MLPs from PVE-HE cells isperformed in the presence of one or more c-myc inhibitors, such asinhibitors of the BET family of bromodomain containing proteins. BETinhibitor may be any molecule or compound that inhibits a BET familymember, and may be a nucleic acid such as DNA and RNA aptamer, antisenseoligonucleotide, siRNA and shRNA, a small peptide, an antibody orantibody fragment, and a small molecule such as a small chemicalcompound. A BET inhibitor may prevent or reduce binding of thebromodomain of at least one BET family member to acetyl-lysine residuesof proteins. It is to be understood that a BET inhibitor may inhibitonly one BET family member or it may inhibit more than one or all BETfamily members.

Examples of BET inhibitors are described in US 2011143651,WO2009/084693A1, WO 2011143669, WO 2011143660, WO 2011054851, and JP2008156311, which are incorporated herein by reference.

Examples of BET inhibitors known in the art include, but are not limitedto, RVX-208 (Resverlogix), PFI-1 (Structural Genomics Consortium),OTX015 (Mitsubishi Tanabe Pharma Corporation), BzT-7, GSK525762A (iBET,GlaxoSmithKline), JQ1 (Cell 2011 146(6):904-17), and the compounds below(WO 2011054851, GlaxoSmithKline):

In some embodiments, the BET inhibitor is a small molecule compound(e.g., JQ1 or derivatives thereof) that binds to the binding pocket ofthe first bromodomain of a BET family member (e.g., BRD1, BRD2, BRD3,BRD4, BRD7, BRDT; see WO 2011143669). Other BET inhibitors include JQ1S,JQ1R, JQ20, JQ8, JQ6, JQ13, JQ19, JQ18, JQ11, JQ21, JQ24B, and KS1.

Another example of a BET inhibitor (referred to herein as iBET) isGSK1210151A (referred to herein as I-BET-151). Other BET inhibitorsinclude IBET151 and IBET762.

Many BET inhibitors are useful as anti-leukemia agents. When used in themethods of this disclosure, they are typically used as lowconcentrations (i.e., below the level at which their cytotoxic effectsare observed).

The disclosure contemplates that the c-myc inhibitors, such as the BETinhibitors, are used during the culture period that differentiates (ormatures) PVE-HE cells to MLP. This culture period usually lasts about 4days. The inhibitor is typically added in the latter half of theculture, including in the last 48 hours, last 36 hours, last 24 hours,last 12 hours, or last 6 hours of the culture.

Other Myc inhibitors include 10058-F4 and CX-3543.

Megakaryocyte Lineage Progenitors

Various embodiments of the present disclosure provide for a method ofgenerating Megakaryocyte Lineage Progenitors (MLPs) from pluripotentstem cells (including human iPS and human ES), as well as compositionsof MLPs.

Early lineage hemogenic endothelial cells are CD41a negative, expressingCD41a during late-stage hemogenic differentiation in hematopoieticprogenitors. CD42b is expressed exclusively in mature megakaryocytes.MLP cultures may be heterogeneous with a high percentage of CD41+ cellsand a low percentage of CD42+ cells.

MLPs may be assessed for the percentage of CD41a and CD42b doublypositive cells by FACS analysis. CD41a is a subunit of fibrinogenreceptor (αIIbβIII) and CD42b is a subunit on von Willebrand Factorreceptor (GPIb-V-IX). The expression of both receptors is specific formegakaryocyte lineages and both are thought to be required for plateletfunction.

Prior to harvesting for cryopreservation MLPs may be assessed for theapproximate percentage of attached cells and the extent ofdifferentiated large cells with low nuclei to cytoplasm ratio. Attachedcells may appear as diffuse colonies with no clear colony borders. Theremay be an abundance of the floating MLPs resting on top of the attachedcell population. Viable floating MLPs may appear clear, with minimalbirefringence, demarked by a smooth cell membrane.

MLPs and compositions thereof may optionally be provided as acryopreserved composition.

In another embodiment, the present disclosure provides for a method ofscreening for a modulator of cellular differentiation comprising:providing a quantity of PVE-HE cells or megakaryocytes progenitors(MLPs); contacting the PVE-HE cells or MLPs with a test compound; anddetermining the presence or absence of a functional effect from thecontact between the PVE-HE cells or MLPs and the test compound, whereinthe presence of a functional effect indicates that the test compound isa megakaryopoietic, thrombopoietic, and/or hematopoietic factor thatmodulates cellular differentiation and the absence of a functionaleffect indicates that the test compound is not a megakaryopoietic,thrombopoietic, and/or hematopoietic factor that modulates cellulardifferentiation. In other embodiments, megakaryopoietic, thrombopoietic,and/or hematopoietic factors relate to the expansion, endomitosis,cytoplasmic maturation, and terminal differentiation of functionalplatelets.

Megakaryocytes

Various embodiments of the present disclosure provide for a method ofgenerating megakaryocytes from pluripotent stem cells (including humaniPS and human ES) under stromal-free conditions and/or under serum-freeconditions. These embodiments include generating megakaryocytes. Furtherembodiments provide for a method of generating megakaryocytes frompluripotent derived hemogenic endothelial cells. Said megakaryocytes arepreferably able to produce platelets, e.g., when cultured under theconditions as described herein.

In one embodiment, the method comprises: providing pluripotent stemcells; and differentiating the pluripotent stem cells intomegakaryocytes. In one embodiment, the pluripotent stem cells are humancells. In another embodiment, the pluripotent stem cells are hESCoptionally produced without the destruction of the embryo such as theNED7 line. In another embodiment, the pluripotent cells are human EScells. In another embodiment, the megakaryocytes are derived frominduced pluripotent stem cells. In another embodiment, the pluripotentstem cells are human iPS cells derived from reprogramming somatic cells.In one embodiment, the somatic cells are from fetal tissue. In anotherembodiment, the somatic cells are from adult tissue.

In another embodiment, the present disclosure provides for a method ofscreening for a modulator of cellular differentiation comprising:providing a quantity of megakaryocytes (MKs); contacting the MKs with atest compound; and determining the presence or absence of a functionaleffect from the contact between the MKs and the test compound, whereinthe presence of a functional effect indicates that the test compound isa megakaryopoietic, thrombopoietic, and/or hematopoietic factor thatmodulates cellular differentiation and the absence of a functionaleffect indicates that the test compound is not a megakaryopoietic,thrombopoietic, and/or hematopoietic factor that modulates cellulardifferentiation. In other embodiments, megakaryopoietic, thrombopoietic,and/or hematopoietic factors relate to the expansion, endomitosis,cytoplasmic maturation, and terminal differentiation of functionalplatelets.

Platelets

Other embodiments of the present disclosure provide for a method ofgenerating platelets from human embryonic stem cells and pluripotentstem cells (including iPSC and human iPSC). In one embodiment, themethod comprises: providing human embryonic stem cells (hESCs); formingPVE-HE cells differentiating the PVE-HE cells into megakaryocytes; anddifferentiating the megakaryocytes into platelets.

In another embodiment, the method of generating platelets comprises:providing PVE-HE cells; differentiating the PVE-HE cells into MLPs ormegakaryocytes; and optionally differentiating the MLPs intomegakaryocytes; and then differentiating (or maturing) themegakaryocytes into platelets, typically through a proplatelet step. Theprocess of differentiating the PVE-HE cells into megakaryocytes can beperformed as described above. In one embodiment, the PVE-HE cells arederived from human ES cells. In another embodiment, the PVE-HE cells arederived from induced pluripotent stem cells (iPSCs). In one embodiment,the iPS cells are human iPS cells derived from reprogramming somaticcells. In one embodiment, the somatic cells are from fetal tissue. Inanother embodiment, the somatic cells are from adult tissue. In variousembodiments, the process of differentiating the megakaryocytes intoplatelets comprises continuing to culture the megakaryocytes to allowthe megakaryocytes to differentiate into platelets. In variousembodiments, the process of differentiating the megakaryocytes intoplatelets is under feeder free conditions and comprises collecting themegakaryocytes differentiated from megakaryocyte-lineage specificprogenitor cells.

In further embodiments, platelets are collected from Day 4 to Day 10 ofmegakaryocyte culture in MK-M medium or other medium comprising Iscove'sModified Dulbecco's Medium (IMDM) as basal medium, human serum albumin,iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, TPO (e.g., at 30 ng/ml), SCF(e.g., at 1 ng/ml), IL-6 (e.g., at 7.5 ng/ml), IL-9 (e.g., at 13.5ng/ml), and optionally a ROCK inhibitor such as Y27632 (e.g., at 5 μM),and/or Heparin (e.g., at 5-25 units/ml). In a preferred embodiment,platelets are collected from 3-5 days after the first emergence ofproplatelet forming cells in the megakaryocyte culture in MK-M medium.In certain embodiments, the platelets are purified using densitygradient centrifugation. In further embodiments, the density gradientcentrifugation uses Percoll medium. In further embodiments, the densitygradient centrifugation uses BSA/HSA medium. In another embodiment, theplatelet purification method separates particles that are CD41anegative. In another embodiment, the platelet purification methodseparates particles that are CD42b negative. In another embodiment, theplatelet purification method retains cell viability and morphologicalintegrity. In other embodiments, the platelets express CD41a and CD42b.In other embodiments, the platelets are responsive to thrombinstimulation. In another embodiment, the platelets are able to spread onfibrinogen and von Willebrand Factor (vWF) surfaces. In additionalembodiments, the platelets have capacity for PAC-1 binding and integrinactivation. In another embodiment, the platelets form micro-aggregatesand facilitate clot formation and retraction. In another embodiment, theplatelets are not activated in the presence of apyrase and/or EDTA.

Various embodiments of the present disclosure provide for a method ofusing pluripotent stem cell-derived platelets. In certain embodiments,the pluripotent stem cell-derived platelets are used in platelettransfusions. The method may comprise providing a quantity ofpluripotent stem cell-derived platelets; and administering the quantityof pluripotent stem cell-derived platelets to a subject in need thereof.In various embodiments the pluripotent stem cell-derived platelets canbe patient-matched platelets. In another embodiment, the platelets arederived from iPSCs. In a certain embodiment, the platelets are derivedfrom human iPS cells. In other embodiments, the platelets are stored ina solution that does not contribute to an HLA alloimmunogenic responsein a subject upon administration of the platelets to the subject. Inadditional exemplary embodiments, the pluripotent stem cell-derivedplatelets may be substantially free of leukocytes, for examplecontaining less than 5%, less than 4%, less than 3%, less than 2%, orless than 1% leukocytes, preferably less than 0.1%, 0.001% or even0.0001%. Additional exemplary embodiments provide a preparation ofpluripotent stem cell-derived platelets which contains less than 10⁶leukocytes in the preparation, more preferably less than 10⁵, 10⁴, oreven 10³ leukocytes.

Additional exemplary embodiments provide a composition containing atleast 10⁸ platelets, more preferably at least 10⁹, at least 10¹⁰, or atleast 10¹¹ platelets.

Using the current blood bank storage conditions at 22-24° C., theviability and function of human platelets (collected by aphaeresis) canbe maintained for only 5 days—the limited storage time though to be dueto aging of the platelets and an increasing risk of bacterialproliferation. It is expected that platelets produced by the methods ofthe present invention will have a longer shelf life than bankedplatelets, such as being able to be maintained for at least 6, 7, 8, 9,10, 11, 12, 13, 14 or even 15 days at 22-24° C. and maintain appropriateviability to be used in human patients.

In certain embodiments, the platelets can be treated—after isolation orduring one or more of the culturing steps leading to plateletproduction—with one or more agents that prolong platelet storage at22-24° C., refrigerated (e.g., 4° C.) and/or frozen.

For example, the present invention contemplates treating the plateletswith agents, or deriving the platelets under conditions, that reducesialidase activity and (optionally) inhibit proliferation of one or morebacteria in a platelet product preparation. The method can include thesteps of contacting the platelet product preparation with an amount of asialidase inhibitor, to thereby obtain a sialidase treated plateletproduct preparation; wherein the sialidase activity is reduced and theproliferation of one or more bacteria is inhibited, as compared to aplatelet product preparation not subjected to a sialidase inhibitor.

The type of bacteria inhibited include those commonly found in plateletproduct preparations. Examples of such bacteria include: Aspergillus,Bacillus sp, Bacteroides eggerthii, Candida albicans, Citrobacter sp,Clostridium perfringens, Corynebacterium sp, Diphtheroid, Enterobacteraerogenes, Enterobacter amnigenus, Enterobacter cloacae, Enterococcusavium, Enterococcus faecalis, Escherichia coli, Fusobacterium spp.,Granulicatella adiacens, Heliobacter pylori, Klebsiella sp, (K.pneumonia, K. oxytoca), Lactobacillus sp, Listeria sp, Micrococcus sp,Peptostreptococcus, Proteus vulgaris, Pseudomonas sp, Pseudomys oxalis,Propionibacterium sp, Salmonella sp, Serratia sp, Serratia marcescensStaphylococcus sp (Coagulase-negative Staphylococcus, Staphylococcusepidermidis, Staphylococcus aureus), Streptococcus sp, (S. gallolyticus,S. bovis, S. pyogenes, S. viridans), and Yersinia enterocolitica.

The sialidase inhibitors that can be used with the present inventioninclude, e.g., fetuin, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid(DANA) or a pharmaceutically acceptable salt thereof; ethyl(3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-1-ene-1-carboxylate);(2R,3R,4S)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylicacid;(4S,5R,6R)-5-acetamido-4-carbamimidamido-6-[(1R,2R)-3-hydroxy-2-methoxypropyl]-5,6-dihydro-4H-pyran-2-carboxylicacid; and(1S,2S,3S,4R)-3-[(1S)-1-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylicacid, or a pharmaceutically acceptable salt thereof.

One or more glycan-modifying agents can be added to the platelets. Suchglycan modifying agents include, for example, CMP-sialic acid, aCMP-sialic acid precursor, UDP galactose or a combination thereof. In anaspect, an enzyme that converts the CMP-sialic acid precursor toCMP-sialic acid can also be added to the platelets.

In certain embodiments, the platelets can be treated with at least oneglycan modifying agent in an amount effective to reduce the clearance ofthe population of platelets. In some embodiments, the glycan modifyingagent is selected from the group consisting UDP-galactose andUDP-galactose precursors. In some preferred embodiments, the glycanmodifying agent is UDP-galactose.

In certain embodiments, the platelets can be treated with certain sugarmolecules which lead to glycation of the exposed GlcNAc residues on GP1band thereby reduced platelet clearance, block platelet phagocytosis,increase platelet circulation time, and/or increase platelet storagetime.

In certain embodiments, the platelets can be treated with trehalose orother low molecular weight polysaccharides.

In certain embodiments, the platelets can be treated with proteaseinhibitors, such as matrix metalloprotease inhibitors.

In some embodiments, the in vivo circulation time of the platelets isincreased by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%,100%, 150%, 200%, or more

The platelets of the present invention can be stored chilled for atleast about 3 days, at least about 5 days, at least about 7 days, atleast about 10 days, at least about 14 days, at least about 21 days, orat least about 28 days.

Further, deriving platelets from stem cells in culture affords theopportunity to precondition the platelets and the megakaryocytes, andeven to genetically modify the stem cells or progenitor cells like themegakaryocytes in a manner that extends the shelf-life of the plateletsand enhances yield and viability after refrigeration and/orcryopreservation storage. For instance, the MKs can be engineered tohave altered levels of expression of genes involved in membrane lipidratios, protein glycosylation patterns, stress-induced proteins, 14-3-3ζtranslocation and the like.

In a further embodiment, the platelets are functional platelets. In afurther embodiment the percentage of functional platelets is at leastabout 60%, is at least about 70%, is at least about 80% or is at leastabout 90%. In a yet further embodiment, the functional platelets areactive for at least 2 days when stored at 22-37° C.

The disclosure further contemplates that the platelets generatedaccording to the methods provided herein can be engineered to includeone or more therapeutic agents which are released by the plateletseither in a passive manner (e.g., they may diffuse out of the plateletover time) or in an active manner (e.g., are released upon activationand degranulation of platelets). A wide range of drugs can be used, andmay include an antibiotic, anti-viral agent, anesthetic, steroidalagent, anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine,antibody, decongestant, antihypertensive, sedative, birth control agent,progestational agent, anti-cholinergic, analgesic, anti-depressant,anti-psychotic, β-adrenergic blocking agent, diuretic, cardiovascularactive agent, vasoactive agent, non-steroidal anti-inflammatory agent,nutritional agent, etc.

For example, the engineered platelets may be prepared so that theyinclude one or more compounds selected from the group consisting ofdrugs that act at synaptic and neuroeffector junctional sites (e.g.,acetylcholine, methacholine, pilocarpine, atropine, scopolamine,physostigmine, succinylcholine, epinephrine, norepinephrine, dopamine,dobutamine, isoproterenol, albuterol, propranolol, serotonin); drugsthat act on the central nervous system (e.g., clonazepam, diazepam,lorazepam, benzocaine, bupivacaine, lidocaine, tetracaine, ropivacaine,amitriptyline, fluoxetine, paroxetine, valproic acid, carbamazepine,bromocriptine, morphine, fentanyl, naltrexone, naloxone); drugs thatmodulate inflammatory responses (e.g., aspirin, indomethacin, ibuprofen,naproxen, steroids, cromolyn sodium, theophylline); drugs that affectrenal and/or cardiovascular function (e.g., furosemide, thiazide,amiloride, spironolactone, captopril, enalapril, lisinopril, diltiazem,nifedipine, verapamil, digoxin, isordil, dobutamine, lidocaine,quinidine, adenosine, digitalis, mevastatin, lovastatin, simvastatin,mevalonate); drugs that affect gastrointestinal function (e.g.,omeprazole, sucralfate); antibiotics (e.g., tetracycline, clindamycin,amphotericin B, quinine, methicillin, vancomycin, penicillin G,amoxicillin, gentamicin, erythromycin, ciprofloxacin, doxycycline,acyclovir, zidovudine (AZT), ddC, ddI, ribavirin, cefaclor, cephalexin,streptomycin, gentamicin, tobramycin, chloramphenicol, isoniazid,fluconazole, amantadine, interferon); anti-cancer agents (e.g.,cyclophosphamide, methotrexate, fluorouracil, cytarabine,mercaptopurine, vinblastine, vincristine, doxorubicin, bleomycin,mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin,decarbazine); immunomodulatory agents (e.g., interleukins, interferons,GM-CSF, TNFα, TNFβ, cyclosporine, FK506, azathioprine, steroids); drugsacting on the blood and/or the blood-forming organs (e.g., interleukins,G-CSF, GM-CSF, erythropoietin, vitamins, iron, copper, vitamin B12,folic acid, heparin, warfarin, coumarin); hormones (e.g., growth hormone(GH), prolactin, luteinizing hormone, TSH, ACTH, insulin, FSH, CG,somatostatin, estrogens, androgens, progesterone, gonadotropin-releasinghormone (GnRH), thyroxine, triiodothyronine); hormone antagonists;agents affecting calcification and bone turnover (e.g., calcium,phosphate, parathyroid hormone (PTH), vitamin D, bisphosphonates,calcitonin, fluoride), vitamins (e.g., riboflavin, nicotinic acid,pyridoxine, pantothenic acid, biotin, choline, inositol, camitine,vitamin C, vitamin A, vitamin E, vitamin K), gene therapy agents (e.g.,viral vectors, nucleic-acid-bearing liposomes, DNA-protein conjugates,anti-sense agents); or other agents such as targeting agents etc.

In certain embodiments, the platelets have been engineered to includeone or more therapeutic agents, such as a small molecule drug, apatameror other nucleic acid agent, or recombinant proteins, i.e., which may bestored in the platelets' granules (α-granules, for example), andpreferably released upon activation of the platelets, such as at thesite of a vascular injury or other wound, atherosclerotic plaque orendothelial cell erosion, infection, or a prothrombotic environmentcapable of platelet activation, such as the vasculature of a solidtumor. In other embodiments, the engineered platelets can be used toreduce the severity of or prevent fibrosis, such as in the treatment oflung fibrosis, i.e., such as may be selected from the group consistingof pulmonary fibrosis, pulmonary hypertension, chronic obstructivepulmonary disease (COPD), asthma and cystic fibrosis.

In certain embodiments, the platelets include one or more exogenousagents which promote or accelerate normal wound healing, reducescarring, reduce fibrosis, or a combination thereof. An exemplaryrecombinant protein which can be expressed in the megakaryocytes andpackaged in the granules of the platelets produced therefrom includeerythropoietin. Localized delivery of erythropoietin acceleratesfibrin-induced wound-healing response, and recombinant EPO-loadedplatelets can be used in treatments of open wounds and sores, includingdiabetic ulcers, burns, etc., as well as closed (internal) woundsincluding surgical procedures (surgical lesion, such as resulting fromlaminectomy, discectomy, joint surgery, abdominal surgery or thoracicsurgery). Other wound-healing proteins, particularly non-fibrotic growthfactors, for which expression in MK cells and storage in plateletgranules can be accomplished according to the present invention includeinsulin-like growth factor 1 (IGF-1); Basic Fibroblast Growth Factor(bFGF); Transforming Growth Factor β-3 (TGFβ-3), granulocyte colonystimulating factor (GCSF), granulocyte macrophage colony stimulatingfactor (GMCSF), keratinocyte growth factor (KGF), fibronectin,vitronectin, thrombospondin, laminin, tenasin.

In certain embodiments, the platelets include one or more exogenousanti-fibrotic agents, such as, but not limited to include antibodies(particularly single chain antibodies) to TGFβ-1, TGFβ-2 and/or PDGF;binding proteins which prevent TGFβ-1, TGFβ-2 and/or PDGF from bindingto their receptors by either binding to the growth factor itself orbinding to the receptor (e.g., peptides containing the receptor bindingsite sequence) or soluble forms of growth factor receptor or the growthfactor binding domains of these receptors; or aptamers which inhibitorreceptor-ligand interaction.

In certain embodiments, the platelets include one or more exogenousagents which modulate wound healing, such as proteases; vasoactivesubstances such as serotonin and/or histamine; fibronectin;collagenases; plasminogen activator; neutral proteases; elastin;collagens; proteogycans; epidermal growth factor (EGF); hormones such asestradiol, testosterone or progesterone; macrophage derived growthfactor (MDGF); adrenomedullin; angiogenin; angiopoietin-1;angiopoitin-related growth factor; brain derived neurotrophic factor;corticotropin-releasing hormone; Cyr16; follistatin; hepatocyte growthfactor; interleukins; midkine; neurokinin A; neuropeptide Y (NPY);pleiotrophin; progranulin, prolifern; secretoneurin; substance P; VG5Q;and factors that recruit pericytes; and becaplermin.

In certain embodiments, the platelets include one or more nucleicapatamers that can promote wound healing and/or reduce fibrosis andscarring at the site of a wound. Scarring is believed to be caused byboth persistent inflammation and overexuberant fibroblast activation.Osteopontin (OPN) is a cytokine that promotes cell activation. Theabsence of OPN in vivo reduces dermal scarring. RNA aptamers are shortRNA molecules that bind target proteins with high affinity. The aptamerOPN-R3 (R3) blocks OPN signaling. In certain embodiments, the plateletscan be loaded with OPN-R3 to be released actively or passively,preferably actively at the site to platelet activation. Exemplary OPNinhibitory apatamers are described in US20110190386.

In certain embodiments, the platelets include one or more small(organic) agents that can promote wound healing and/or reduce fibrosisand scarring at the site of a wound. Excisional wound closure, forexample, can be significantly accelerated by A2A receptor agonists, suchas CGS-21680 (Montesinos et al. JEM 1997, 186(9) pages 1615-1620).Accordingly, merely to illustrate, the platelets can be loaded with anA2A receptor agonists to be released actively or passively, preferablyactively at the site to platelet activation. Other small molecule agentsinclude steroids, nonsteroidal antiinflammatory compounds (NSAID),5-lipoxygenase (5-LO) inhibitors, leukotriene B4 (LTB4) receptorantagonists, leukotriene A4 (LTA4) hydrolase inhibitors, 5-HT agonists,3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) inhibitors, H2antagonists, antineoplastic agents, and cyclooxygenase-2 inhibitors.

In certain embodiments, the platelets include one or more exogenousantibiotic agents. Exemplary antibiotics include chloramphenicol,chlortetracycline, clyndamycin, clioquinol, erythromycin, framycetin,gramicidin, fusidic acid, gentamicin, mafenide, mupiroicin, neomycin,polymyxin B, bacitracin, silver sulfadiazine, tetracycline,chlortetracycline, tobramycin, amikacin, vancomycin, ramoplanin,levofloxacin, ofloxacin, moxifloxacin, clindamycin or combinationsthereof.

In certain embodiments, the platelets include one or more exogenousanalgesic or anesthetic and/or anti-inflammatory agents. Exemplaryanti-inflammatory agents can be selected from acetaminophen, aspirin,ibuprofen, diclofenac, indometacin, piroxicam, fenoprofen, flubiprofen,ketoprofen, naproxen, suprofen, loxoprofen, cinnoxicam, tenoxicam, and acombination thereof.

Platelets engineered to deliver antithrombotic/antirestenosis agents canbe used during angioplasty and thrombolysis procedures.

In certain embodiments, the engineered platelets can be used to preventor reduce the severity of atherosclerosis, and may include one or moreexogenous anti-atherosclerosis agent (i.e., an agent that reducesatherosclerotic lesions or prevents formation), and may include:antiproliferative/antimitotic agents including natural products such asvinca alkaloids (i.e. vinblastine, vincristine, and vinorelbine),paclitaxel, epidipodophyllotoxins (i.e. etoposide, teniposide),antibiotics (dactinomycin (actinomycin D) daunorubicin, doxorubicin andidarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin(mithramycin) and mitomycin, enzymes (L-asparaginase which systemicallymetabolizes L-asparagine and deprives cells which do not have thecapacity to synthesize their own asparagine);antiproliferative/antimitotic alkylating agents such as nitrogenmustards (mechlorethamine, cyclophosphamide and analogs, melphalan,chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine andthiotepa), alkyl sulfonates-busulfan, nirtosoureas (carmustine (BCNU)and analogs, streptozocin), trazenes-dacarbazinine (DTIC);anti-proliferative/antimitotic antimetabolites such as folic acidanalogs (methotrexate), pyrimidine analogs (fluorouracil, floxuridine,and cytarabine), purine analogs and related inhibitors (mercaptopurine,thioguanine, pentostatin and 2-chlorodeoxyadenosine {cladribine });platinum coordination complexes (cisplatin, carboplatin), procarbazine,hydroxyurea, mitotane, aminoglutethimide; hormones (i.e. estrogen);fibrinolytic agents (such as tissue plasminogen activator, streptokinaseand urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel,abciximab; antimigratory; antisecretory (breveldin); anti-inflammatory:such as adrenocortical steroids (Cortisol, cortisone, fludrocortisone,prednisone, prednisolone, 6a-methylprednisolone, triamcinolone,betamethasone, and dexamethasone), non-steroidal agents (salicylic acidderivatives i.e. aspirin; para-aminophenol derivatives i.e.acetaminophen; indole and indene acetic acids (indomethacin, sulindac,and etodalac), heteroaryl acetic acids (tolmetin, diclofenac, andketorolac), arylpropionic acids (ibuprofen and derivatives), anthranilicacids (mefenamic acid, and meclofenamic acid), enolic acids (piroxicam,tenoxicam, phenylbutazone, and oxyphenthatrazone), nabumetone, goldcompounds (auranofin, aurothioglucose, gold sodium thiomalate);immunosuppressives: (cyclosporine, tacrolimus (FK-506), sirolimus(rapamycin), azathioprine, mycophenolate mofetil); angiogenic agents:vascular endothelial growth factor (VEGF), fibroblast growth factor(FGF); angiotensin receptor blockers; nitric oxide donors; antisenseoligionucleotides and combinations thereof; cell cycle inhibitors, mTORinhibitors, and growth factor receptor signal transduction kinaseinhibitors; retenoids; cyclin/CDK inhibitors; HMG co-enzyme reductaseinhibitors (statins); and protease inhibitors.

In certain embodiments, the engineered platelets can used to prevent orreduce the severity of restenosis, and may include one or more exogenousanti-antiproliferative substances, antiphlogistic as well asantithrombotic compounds are used as active agents. Exemplary activeactive agents for restenosis include sirolimus, everolimus,somatostatin, tacrolimus, roxithromycin, dunaimycin, ascomycin,bafilomycin, erythromycin, midecamycin, josamycin, concanamycin,clarithromycin, troleandomycin, folimycin, cerivastatin, simvastatin,lovastatin, fluvastatin, rosuvastatin, atorvastatin, pravastatin,pitavastatin, vinblastine, vincristine, vindesine, vinorelbine,etoboside, teniposide, nimustine, carmustine, lomustine,cyclophosphamide, 4-hydroxyoxycyclophosphamide, estramustine, melphalan,ifosfamide, tropfosfamide, chlorambucil, bendamustine, dacarbazine,busulfan, procarbazine, treosulfan, tremozolomide, thiotepa,daunorubicin, doxorubicin, aclarubicin, epirubicin, mitoxantrone,idarubicin, bleomycin, mitomycin, dactinomycin, methotrexate,fludarabine, fludarabine-5′-dihydrogenphosphate, cladribine,mercaptopurine, thioguanine, cytarabine, fluorouracil, gemcitabine,capecitabine, docetaxel, carboplatin, cisplatin, oxaliplatin, amsacrine,irinotecan, topotecan, hydroxycarbamide, miltefosine, pentostatin,aldesleukin, tretinoin, asparaginase, pegasparase, anastrozole,exemestane, letrozole, formestane, aminoglutethemide, adriamycin,azithromycin, spiramycin, cepharantin, smc proliferation inhibitor-2w,epothilone A and B, mitoxantrone, azathioprine, mycophenolatmofetil,c-myc-antisense, b-myc-antisense, betulinic acid, camptothecin,lapachol, β-lapachone, podophyllotoxin, betulin, podophyllic acid2-ethylhydrazide, molgramostim, peginterferon α-2b, lenograstim;filgrastim, macrogol, dacarbazine, basiliximab, daclizumab, selectin,cytokine antagonist, CETP inhibitor, cadherines, cytokinin inhibitors,COX-2 inhibitor, NFκ.B, angiopeptin, ciprofloxacin, camptothecin,fluroblastin, monoclonal antibodies, which inhibit the muscle cellproliferation, bFGF antagonists, probucol, prostaglandins,1,11-dimethoxycanthin-6-one, 1-hydroxy-11-methoxycanthin-6-one,scopolectin, colchicine, NO donors, pentaerythritol tetranitrate,syndnoeimines, S-nitrosoderivatives, tamoxifen, staurosporine,β-estradiol, α-estradiol, estriol, estrone, ethinylestradiol,fosfestrol, medroxyprogesterone, estradiol cypionates, estradiolbenzoates, tranilast, kamebakaurin and other terpenoids, which areapplied in the therapy of cancer, verapamil, tyrosine kinase inhibitors,tyrphostines, cyclosporine A, paclitaxel and derivatives thereof,baccatin, taxotere and other both synthetically and from native sourcesobtained macrocyclic oligomers of carbon suboxide (MCS) and derivativesthereof, mofebutazone, acemetacin, diclofenac, lonazolac, dapsone,o-carbamoylphenoxyacetic acid, lidocaine, ketoprofen, mefenamic acid,piroxicam, meloxicam, chloroquine phosphate, penicillamine,hydroxychloroquine, auranofin, sodium aurothiomalate, oxaceprol,celecoxib, β-sitosterin, ademetionine, myrtecaine, polidocanol,nonivamide, levomenthol, benzocaine, aescin, ellipticine, CalbiochemD-24851, colcemid, cytochalasin A-E, indanocine, nocadazole, S 100protein, bacitracin, vitronectin receptor antagonists, azelastine,guanidyl cyclase stimulator tissue inhibitor of metal proteinase-1 and-2, free nucleic acids, nucleic acids incorporated into virustransmitters, DNA and RNA fragments, plaminogen activator inhibitor-1,plasminogen activator inhibitor-2, antisense oligonucleotides, VEGFinhibitors, IGF-1, antibiotics, antithrombotics, argatroban, aspirin,abciximab, synthetic antithrombin, bivalirudin, coumadin, enoxoparin,desulphated and N-reacetylated heparin, tissue plasminogen activator,GpIIb/IIIa platelet membrane receptor, factor X a inhibitor antibody,hirudin, r-hirudin, PPACK, protamin, prourokinase, streptokinase,warfarin, urokinase, vasodilators, dipyramidole, trapidil,nitroprussides, PDGF antagonists, triazolopyrimidine and seramin, ACEinhibitors, captopril, cilazapril, lisinopril, enalapril, losartan,thioprotease inhibitors, prostacyclin, vapiprost, interferon α, β and γ,histamine antagonists, serotonin blockers, apoptosis inhibitors,apoptosis regulators, p65 NF-κ.B and Bcl-xL antisense oligonucleotides,halofuginone, nifedipine, tocopherol, tranirast, molsidomine, teapolyphenols, epicatechin gallate, epigallocatechin gallate, Boswellicacids and derivatives thereof, leflunomide, anakinra, etanercept,sulfasalazine, etoposide, dicloxacillin, tetracycline, triamcinolone,mutamycin, procainimid, retinoic acid, quinidine, disopyrimide,flecainide, propafenone, sotolol, amidorone, natural and syntheticallyobtained steroids, bryophyllin A, inotodiol, maquiroside A,ghalakinoside, mansonine, strebloside, hydrocortisone, betamethasone,dexamethasone, fenoporfen, ibuprofen, indomethacin, naproxen,phenylbutazone, antiviral agents, antimycotics, antiprozoal agents,natural terpenoids, hippocaesculin, barringtogenol-C21-angelate,14-dehydroagrostistachin, agroskerin, agrostistachin,17-hydroxyagrostistachin, ovatodiolids, 4,7-oxycycloanisomelic acid,baccharinoids B1, B2, B3 and B7, tubeimoside, bruceanol A, B and C,bruceantinoside C, yadanziosides N and P, isodeoxyelephantopin,tomenphantopin A and B, coronarin A, B, C and D, ursolic acid, hyptaticacid A, zeorin, iso-iridogermanal, maytenfoliol, effusantin A, excisaninA and B, longikaurin B, sculponeatin C, kamebaunin, leukamenin A and B,13,18-dehydro-6-α-senecioyloxychaparrin, taxamairin A and B, regenilol,triptolide, cymarin, apocymarin, aristolochic acid, anopterin,hydroxyanopterin, anemonin, protoanemonin, berberine, cheliburinchloride, cictoxin, sinococuline, bombrestatin A and B, cudraisoflavoneA, curcumin, dihydronitidine, nitidine chloride,12-β-hydroxypregnadien-3,20-dione, bilobol, ginkgol, ginkgolic acid,helenalin, indicine, indicine-N-oxide, lasiocarpine, inotodiol,glycoside 1a, podophyllotoxin, justicidin A and B, larreatin,malloterin, mallotochromanol, isobutyrylmallotochromanol, maquiroside A,marchantin A, maytansine, lycoridicin, margetine, pancratistatin,liriodenine, bisparthenolidine, oxoushinsunine, aristolactam-AII,bisparthenolidine, periplocoside A, ghalakinoside, ursolic acid,deoxypsorospermin, psycorubin, ricin A, sanguinarine, manwu wheat acid,methylsorbifolin, sphatheliachromen, stizophyllin, mansonine,strebloside, akagerine, dihydrousambaraensine, hydroxyusambarine,strychnopentamine, strychnophylline, usambarine, usambarensine,berberine, liriodenine, oxoushinsunine, daphnoretin, lariciresinol,methoxylariciresinol, syringaresinol, umbelliferon, afromoson,acetylvismione B, desacetylvismione A, or vismione A and B.

In still other embodiments, the engineered platelets can used as part ofa treatment for solid tumors. Solid tumors generate a prothromboticenvironment capable of platelet activation. Recent findings indicatethat the activated platelets are crucial regulators of tumor vascularhomeostasis in that they prevent tumor hemorrhage. Surprisingly, thiseffect is independent of platelets' capacity to form thrombi and insteadrelies on the secretion of their granule content. Thus, using plateletsecretory activities to target the release of anti-tumor and/oranti-angiogeneic agents represents an approach to specifically killtumor cells and/or destabilize tumor vasculature. In certain preferredembodiments, the engineered platelets can be loaded withanti-angiogeneic agents and/or agents causing the disruption of tumorvascular structure.

To further illustrate, the engineered platelets can be loaded with suchanticancer agents as anti-neoplastic or chemotherapeutic agents, whichmay include (a) alkylating agents, such as mechlorethamine,cyclophosphamide, ifosfamide, melphaan, chlorambucil,hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine,semustine, streptozocin, dacarbazine, etc.; (b) antimetabolites, such asmethotrexate, 5-FU, FudR, cytarabine, 6 MP, thioguanine, pentostatin,etc.; (c) natural products, such as taxol, vinblastine, vincristine,etoposide, teniposide, etc.; (d) antibiotics such as dactinomycin,daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin c, etc.; (e)enzymes such as L-asparaginase, heparinases, chondroitinases, etc.; (f)interferons and interleukins, such as interferon-α, interferon-γ, tumornecrosis factor, etc.; (g) platinum coordination complexes such ascisplatin, carboplatin or their derivatives; and (h) other miscellaneousagents such as mitoxantrone, bischloroethyl nitrosourea, hydroxyurea,chloroethyl-cyclohexyl nitrosourea, prednisone, diethylstilbestrol,medroxyprogesterone, tamoxifen, mitotane, procarbazine,aminoglutethimide, progestins, androgens, antiadrogens, Leuprolide, etc.

In an exemplary embodiment, the engineered platelets include arecombinant protein which acts as an inhibitor of VEGF (i.e., VEGFantagonists). Such proteins include antibodies and antibody analogs(such as single chain antibodies, monobodies, antigen binding sites andthe like) such as ranibizumab, VEGF-traps such as Aflibercept which aresoluble proteins including ligand binding domains from VEGF receptors,which bind to either VEGF or the VEGF receptor and block receptoractivation. In preferred embodiments, the polypeptide VEGF antagonist isexpressed in MK cells in a manner in which it is incorporated intogranules, particularly α-granules, of the platelets and released uponactivation of the platelets.

Although, a preferred use of the engineered platelets composition wouldbe in tumor therapy, both solid and myeloid, the same principle isembodied in the treatment of other abnormal angiogenesis-basedpathologies. Other pathologies may include arthritis, retinopathies,psoriasis, solid tumors, benign tumors, Kaposi's sarcoma, andhematological malignancies. This could include drugs described earlier;or for example in the case of arthritis, it may comprise of diseasemodifying drugs (DMARDs), non-steroidal anti-inflammatory drugs(NSAIDS), Colchicine, methotrexate, etc.

In exemplary embodiments, the engineered platelets can be loaded withsuch anticancer drugs as may be selected from acivicin, aclarubicin,acodazole, acronycine, adozelesin, alanosine, aldesleukin, allopurinolsodium, altretamine, aminoglutethimide, amonafide, ampligen, amsacrine,androgens, anguidine, aphidicolin glycinate, asaley, asparaginase,5-azacitidine, azathioprine, Bacillus calmette-guerin (BCG), Baker'sAntifol (soluble), beta-2′-deoxythioguanosine, bisantrene HCl, bleomycinsulfate, busulfan, buthionine sulfoximine, BWA 773U82, BW 502U83.HCl, BW7U85 mesylate, ceracemide, carbetimer, carboplatin, carmustine,chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycinA3, cisplatin, cladribine, corticosteroids, Corynebacterium parvum,CPT-11, crisnatol, cyclocytidine, cyclophosphamide, cytarabine,cytembena, dabis maleate, dacarbazine, dactinomycin, daunorubicin HCl,deazauridine, dexrazoxane, dianhydrogalactitol, diaziquone,dibromodulcitol, didemnin B, diethyldithiocarbamate, diglycoaldehyde,dihydro-5-azacytidine, doxorubicin, echinomycin, edatrexate, edelfosine,eflornithine, Elliott's solution, elsamitrucin, epirubicin, esorubicin,estramustine phosphate, estrogens, etanidazole, ethiofos, etoposide,fadrazole, fazarabine, fenretinide, filgrastim, finasteride, flavoneacetic acid, floxuridine, fludarabine phosphate, 5-fluorouracil,Fluosol., flutamide, gallium nitrate, gemcitabine, goserelin acetate,hepsulfam, hexamethylene bisacetamide, homoharringtonine, hydrazinesulfate, 4-hydroxyandrostenedione, hydrozyurea, idarubicin HCl,ifosfamide, interferon alfa, interferon beta, interferon gamma,interleukin-1 alpha and beta, interleukin-3, interleukin-4,interleukin-6, 4-ipomeanol, iproplatin, isotretinoin, leucovorincalcium, leuprolide acetate, levamisole, liposomal daunorubicin,liposome encapsulated doxorubicin, lomustine, lonidamine, maytansine,mechlorethamine hydrochloride, melphalan, menogaril, merbarone,6-mercaptopurine, mesna, methanol extraction residue of Bacilluscalmette-guerin, methotrexate, N-methylformamide, mifepristone,mitoguazone, mitomycin-C, mitotane, mitoxantrone hydrochloride,monocyte/macrophage colony-stimulating factor, nabilone, nafoxidine,neocarzinostatin, octreotide acetate, ormaplatin, oxaliplatin,paclitaxel, pala, pentostatin, piperazinedione, pipobroman, pirarubicin,piritrexim, piroxantrone hydrochloride, PIXY-321, plicamycin, porfimersodium, prednimustine, procarbazine, progestins, pyrazofurin, razoxane,sargramostim, semustine, spirogermanium, spiromustine, streptonigrin,streptozocin, sulofenur, suramin sodium, tamoxifen, taxotere, tegafur,teniposide, terephthalamidine, teroxirone, thioguanine, thiotepa,thymidine injection, tiazofurin, topotecan, toremifene, tretinoin,trifluoperazine hydrochloride, trifluridine, trimetrexate, tumornecrosis factor, uracil mustard, vinblastine sulfate, vincristinesulfate, vindesine, vinorelbine, vinzolidine, Yoshi 864, zorubicin, andmixtures thereof.

The engineered platelets may include one or more immunostimulatoryagents in order to promote immune activation against tumor cells, so mayinclude such agents as a toll like receptor (TLR) agonist, TLR4, TLR7,TLR9, N-acetylmuramyl-L-alanine-D-isoglutamine (MDP),lipopolysaccharides (LPS), genetically modified and/or degraded LPS,alum, glucan, colony stimulating factors, EPO, GM-CSF, G-CSF, M-CSF,pegylated G-CSF, SCF, IL-3, IL6, PIXY 321, interferons, γ-interferon,α-interferon, interleukins, IL-2, IL-7, IL-12, IL-15, IL-18, MHC ClassII binding peptides, saponins, QS2I, unmethylated CpG sequences,I-methyl tryptophan, arginase inhibitors, cyclophosphamide, orantibodies that block immunosuppressive functions, anti-CTLA4antibodies, or mixtures of two or more thereof.

In certain instances, particular for creating engineered plateletsincluding small molecule drugs and/or nucleic acids, the active agent(s)can be introduced into the platelets by addition to the culture mediafor megakaryocytes, proplatelets or other cells along thedifferentiation pathway, or can be provided in media/solutions in whichthe platelets are incubated.

In other instances, particular for creating engineered plateletsincluding protein therapeutics, the active agent(s) can be recombinantlyexpressed by the megakaryocytes, proplatelets or other cells along thedifferentiation pathway, and can thus be present in the resultingplatelets. In preferred embodiments, the recombinant protein is packagedin the platelet granules (especially α-granules). Certain recombinantproteins are automatically trafficked into the granules, such as FactorVIII, otherwise may require the use of fusion proteins which includegranule targeting moieties which traffic the fusion protein to plateletgranules. An exemplary granule targeting moiety is platelet factor 4(PF4), or a portion thereof sufficient to traffic the resulting fusionprotein to platelet granules. See, for example, Briguet-Laugier et al.,J Thromb Haemost. 2004 2(12):2231-40; El Goli et al. J Biol Chem. 2005280(34):30329-35;

An exemplary embodiment of a recombinant protein that requires noaddition granule trafficking moiety is Factor VIII. The presentinvention contemplates platelets that have been engineered to haveFactor VIII stored in their a-granules, and which release therecombinant protein upon activation such as at the site of a wound.Hemophilia A is an X chromosome-linked bleeding disorder caused bydefects in the factor VIII (FVIII) gene and affecting approximately1:5000 male individuals. Current treatment consists of factorreplacement by using pooled FVIII concentrate or recombinant product.The limitations of these products include their expense and the limitedability of these products to prevent long-term sequelae unless used in arigorous prophylaxis regimen. About 10% of the population withhemophilia A develops inhibitors to the infusion product, requiringalternative, even more expensive and less effective, forms of therapy.Concerns about infectious complications from blood-derived replacementproducts have continued to be an issue even with new preparativetechniques. The high costs of treatment, the infectious and immunecomplications of therapy, and the limitations in preventing thelong-term complications of hemophilia A make a platelet deliverystrategy for the treatment of hemophilia A an attractive alternate formof therapy.

To further illustrate, Yarovoi et al. Blood, 2003 102(12): 4007describes an expression construct that can be used to generate theFactor VIII engineered platelets of the present invention. Inparticular, the coding sequence for (human) Factor VIII can be placedunder the regulatory control of the megakaryocyte-specific glycoproteinIb (GPIbα) proximal promoter region. See Fujita et al. Blood. 1998;92:488-495. The ectopically expressed factor VIII in developingmegakaryocytes is stored in α-granules, which are contained within theplatelets produced by the MKs, and then released from circulatingplatelets upon activation.

Pharmaceutical Preparations

Exemplary compositions of the present disclosure may be formulationsuitable for use in treating a human patient, such as pyrogen-free oressentially pyrogen-free, and pathogen-free. When administered, thepharmaceutical preparations for use in this disclosure may be in apyrogen-free, pathogen-free, physiologically acceptable form.

Additional exemplary compositions of the present disclosure may beirradiated, e.g., prior to administration. For example, the cells may beirradiated with gamma irradiation, e.g., with a dosage of approximately25 gy. For example, the composition may be irradiated with a dosagesufficient to mitotically inactivate any nucleated eukaryotic cells,and/or pathogens contained in the composition, e.g., in a dosagesufficient to mitotically inactivate any pluripotent stem cells, MKs,leukocytes, and/or PVE-HE that may be contained therein.

Delivery of Platelets

Various membranes, devices and methods of their manufacture have beenproposed and evaluated as a means of transplanting cells and theirsecreted products into the human body and are collectively referred toin the patent literature as bioartificial implants. Typically they sharea common principle of operation, that is, the cells are sequesteredinside a chamber bounded by a semipermeable membrane. Long term cellviability is thought to rely on the sustained diffusive exchange ofnutrients and waste products with adjacent vascularized tissue. (U.S.Pat. Nos. 6,372,244, 6,113,938, 6,322,804 4,911,717, 5,855,613,6,083,523, 5,916,554, 6,511,473, 6,485,723). There are three major typesof devices described in the scientific and patent literature forimplantation of cells into various tissue compartments including: planardisk designs, hollow fiber-based designs, and geometric solid-baseddesigns. These devices are typically designed to be placed in a bodycavity.

Definitions

“Embryoid bodies” refers to clumps or clusters of pluripotent cells(e.g., iPSC or ESC) which may be formed by culturing pluripotent cellsunder non-attached conditions, e.g., on a low-adherent substrate or in a“hanging drop.” In these cultures, pluripotent cells can form clumps orclusters of cells denominated as embryoid bodies. See Itskovitz-Eldor etal., Mol Med. 2000 February; 6(2):88-95, which is hereby incorporated byreference in its entirety. Typically, embryoid bodies initially form assolid clumps or clusters of pluripotent cells, and over time some of theembryoid bodies come to include fluid filled cavities, the latter formerbeing referred to in the literature as “simple” EBs and the latter as“cystic” embryoid bodies.

The term “embryonic stem cells” (ES cells) is used herein as it is usedin the art. This term includes cells derived from the inner cell mass ofhuman blastocysts or morulae, including those that have been seriallypassaged as cell lines. The ES cells may be derived from fertilizationof an egg cell with sperm, as well as using DNA, nuclear transfer,parthenogenesis, or by means to generate ES cells with homozygosity inthe HLA region. ES cells are also cells derived from a zygote,blastomeres, or blastocyst-staged mammalian embryo produced by thefusion of a sperm and egg cell, nuclear transfer, parthenogenesis,androgenesis, or the reprogramming of chromatin and subsequentincorporation of the reprogrammed chromatin into a plasma membrane toproduce a cell. Embryonic stem cells, regardless of their source or theparticular method used to produce them, can be identified based on (i)the ability to differentiate into cells of all three germ layers, (ii)expression of at least Oct 4 and alkaline phosphatase, and (iii) abilityto produce teratomas when transplanted into immunodeficient animals.Embryonic stem cells that may be used in embodiments of the presentinvention include, but are not limited to, human ES cells (“ESC” or “hEScells”) such as MA01, MA09, ACT-4, No. 3, H1, H7, H9, H14 and ACT30embryonic stem cells. Additional exemplary cell lines include NED1,NED2, NED3, NED4, NED5, and NED7. See also NIH Human Embryonic Stem CellRegistry. An exemplary human embryonic stem cell line that may be usedis MA09 cells. The isolation and preparation of MA09 cells waspreviously described in Klimanskaya, et al. (2006) “Human Embryonic StemCell lines Derived from Single Blastomeres.” Nature 444: 481-485. Theisolation and preparation of other ES cells that can be used inaccordance with this disclosure was also previously described in Chunget al. (2008) “Human Embryonic Stem Cell Line Generated Without EmbryoDestruction”, Cell Stem Cell, 2:113-117. The human ES cells used inaccordance with exemplary embodiments of the present invention may bederived and maintained in accordance with GMP standards.

As used herein, the term “pluripotent stem cells” includes embryonicstem cells, embryo-derived stem cells, and induced pluripotent stemcells and other stem cells having the capacity to form cells from allthree germ layers of the body, regardless of the method by which thepluripotent stem cells are derived. Pluripotent stem cells are definedfunctionally as stem cells that are: (a) capable of inducing teratomaswhen transplanted in immunodeficient (SCID) mice; (b) capable ofdifferentiating to cell types of all three germ layers (e.g., candifferentiate to ectodermal, mesodermal, and endodermal cell types); and(c) express one or more markers of embryonic stem cells (e.g., expressOct 4, alkaline phosphatase. SSEA-3 surface antigen, SSEA-4 surfaceantigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). In certainembodiments, pluripotent stem cells express one or more markers selectedfrom the group consisting of: OCT-4, alkaline phosphatase, SSEA-3,SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can begenerated using, for example, methods known in the art. Exemplarypluripotent stem cells include embryonic stem cells derived from the ICMof blastocyst stage embryos, as well as embryonic stem cells derivedfrom one or more blastomeres of a cleavage stage or morula stage embryo(optionally without destroying the remainder of the embryo). Suchembryonic stem cells can be generated from embryonic material producedby fertilization or by asexual means, including somatic cell nucleartransfer (SCNT), parthenogenesis, and androgenesis. Further exemplarypluripotent stem cells include induced pluripotent stem cells (iPSCs)generated by reprogramming a somatic cell by expressing a combination offactors (herein referred to as reprogramming factors). The iPSCs can begenerated using fetal, postnatal, newborn, juvenile, or adult somaticcells.

In certain embodiments, factors that can be used to reprogram somaticcells to pluripotent stem cells include, for example, a combination ofOct 4 (sometimes referred to as Oct 3/4), Sox2, c-Myc, and Klf4. Inother embodiments, factors that can be used to reprogram somatic cellsto pluripotent stem cells include, for example, a combination of Oct 4,Sox2, Nanog, and Lin28. In certain embodiments, at least tworeprogramming factors are expressed in a somatic cell to successfullyreprogram the somatic cell. In other embodiments, at least threereprogramming factors are expressed in a somatic cell to successfullyreprogram the somatic cell. In other embodiments, at least fourreprogramming factors are expressed in a somatic cell to successfullyreprogram the somatic cell. In other embodiments, additionalreprogramming factors are identified and used alone or in combinationwith one or more known reprogramming factors to reprogram a somatic cellto a pluripotent stem cell. Induced pluripotent stem cells are definedfunctionally and include cells that are reprogrammed using any of avariety of methods (integrative vectors, non-integrative vectors,chemical means, etc.). Pluripotent stem cells may be geneticallymodified or otherwise modified to increase longevity, potency, homing,to prevent or reduce alloimmune responses or to deliver a desired factorin cells that are differentiated from such pluripotent cells (forexample, platelets).

“Induced pluripotent stem cells” (iPS cells or iPSC) can be produced byprotein transduction of reprogramming factors in a somatic cell. Incertain embodiments, at least two reprogramming proteins are transducedinto a somatic cell to successfully reprogram the somatic cell. In otherembodiments, at least three reprogramming proteins are transduced into asomatic cell to successfully reprogram the somatic cell. In otherembodiments, at least four reprogramming proteins are transduced into asomatic cell to successfully reprogram the somatic cell.

The pluripotent stem cells can be from any species. Embryonic stem cellshave been successfully derived in, for example, mice, multiple speciesof non-human primates, and humans, and embryonic stem-like cells havebeen generated from numerous additional species. Thus, one of skill inthe art can generate embryonic stem cells and embryo-derived stem cellsfrom any species, including but not limited to, human, non-humanprimates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs(domestic and wild dogs), cats (domestic and wild cats such as lions,tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig,goats, elephants, panda (including giant panda), pigs, raccoon, horse,zebra, marine mammals (dolphin, whales, etc.) and the like. In certainembodiments, the species is an endangered species. In certainembodiments, the species is a currently extinct species.

Similarly, iPS cells can be from any species. These iPS cells have beensuccessfully generated using mouse and human cells. Furthermore, iPScells have been successfully generated using embryonic, fetal, newborn,and adult tissue. Accordingly, one can readily generate iPS cells usinga donor cell from any species. Thus, one can generate iPS cells from anyspecies, including but not limited to, human, non-human primates,rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic andwild dogs), cats (domestic and wild cats such as lions, tigers,cheetahs), rabbits, hamsters, goats, elephants, panda (including giantpanda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales,etc.) and the like. In certain embodiments, the species is an endangeredspecies. In certain embodiments, the species is a currently extinctspecies.

Induced pluripotent stem cells can be generated using, as a startingpoint, virtually any somatic cell of any developmental stage. Forexample, the cell can be from an embryo, fetus, neonate, juvenile, oradult donor. Exemplary somatic cells that can be used includefibroblasts, such as dermal fibroblasts obtained by a skin sample orbiopsy, synoviocytes from synovial tissue, foreskin cells, cheek cells,or lung fibroblasts. Although skin and cheek provide a readily availableand easily attainable source of appropriate cells, virtually any cellcan be used. In certain embodiments, the somatic cell is not afibroblast.

The induced pluripotent stem cell may be produced by expressing orinducing the expression of one or more reprogramming factors in asomatic cell. The somatic cell may be a fibroblast, such as a dermalfibroblast, synovial fibroblast, or lung fibroblast, or anon-fibroblastic somatic cell. The somatic cell may be reprogrammedthrough causing expression of (such as through viral transduction,integrating or non-integrating vectors, etc.) and/or contact with (e.g.,using protein transduction domains, electroporation, microinjection,cationic amphiphiles, fusion with lipid bilayers containing, detergentpermeabilization, etc.) at least 1, 2, 3, 4, 5 reprogramming factors.The reprogramming factors may be selected from Oct 3/4, Sox2, NANOG,Lin28, c Myc, and Klf4. Expression of the reprogramming factors may beinduced by contacting the somatic cells with at least one agent, such asa small organic molecule agents, that induce expression of reprogrammingfactors.

Further exemplary pluripotent stem cells include induced pluripotentstem cells generated by reprogramming a somatic cell by expressing orinducing expression of a combination of factors (“reprogrammingfactors”). iPS cells may be obtained from a cell bank. The making of iPScells may be an initial step in the production of differentiated cells.iPS cells may be specifically generated using material from a particularpatient or matched donor with the goal of generating tissue-matchedmegakaryocytes and platelets. iPSCs can be produced from cells that arenot substantially immunogenic in an intended recipient, e.g., producedfrom autologous cells or from cells histocompatible to an intendedrecipient.

The somatic cell may also be reprogrammed using a combinatorial approachwherein the reprogramming factor is expressed (e.g., using a viralvector, plasmid, and the like) and the expression of the reprogrammingfactor is induced (e.g., using a small organic molecule.) For example,reprogramming factors may be expressed in the somatic cell by infectionusing a viral vector, such as a retroviral vector or a lentiviralvector. Also, reprogramming factors may be expressed in the somatic cellusing a non-integrative vector, such as an episomal plasmid. See, e.g.,Yu et al., Science. 2009 May 8; 324(5928):797-801, which is herebyincorporated by reference in its entirety. When reprogramming factorsare expressed using non-integrative vectors, the factors may beexpressed in the cells using electroporation, transfection, ortransformation of the somatic cells with the vectors. For example, inmouse cells, expression of four factors (Oct3/4, Sox2, c myc, and Klf4)using integrative viral vectors is sufficient to reprogram a somaticcell. In human cells, expression of four factors (Oct3/4, Sox2, NANOG,and Lin28) using integrative viral vectors is sufficient to reprogram asomatic cell.

Once the reprogramming factors are expressed in the cells, the cells maybe cultured. Over time, cells with ES characteristics appear in theculture dish. The cells may be chosen and subcultured based on, forexample, ES morphology, or based on expression of a selectable ordetectable marker. The cells may be cultured to produce a culture ofcells that resemble ES cells—these are putative iPS cells.

To confirm the pluripotency of the iPS cells, the cells may be tested inone or more assays of pluripotency. For example, the cells may be testedfor expression of ES cell markers; the cells may be evaluated forability to produce teratomas when transplanted into SCID mice; the cellsmay be evaluated for ability to differentiate to produce cell types ofall three germ layers. Once a pluripotent iPSC is obtained it may beused to produce megakaryocyte cells and platelets.

The term “hemogenic endothelial cells” (PVE-HE) as used herein refers tocells capable of differentiating to give rise to hematopoietic celltypes or endothelial cell types, which may express PECAM1, VE-Cadherin,and/or endoglin (e.g., PECAM1+VE-Cad+Endoglin+hemogenic PVE-HE), andwhich may optionally be derived from pluripotent stem cells. These cellscan be described based on numerous structural and functional propertiesincluding, but not limited to, expression (RNA or protein) or lack ofexpression (RNA or protein) of one or more markers. The PVE-HE cells arecharacterized by the expression of the markers CD31 (PECAM1). Forexample, at least about 90%, at least about 95%, or at least about 9addition, immunofluorescence and transmission electron microscopicresults further demonstrate 9% of the PVE-HE cells in a population maybe CD31+. The PVE-HE cells may also express the markers CD105(endoglin), and CD144 (VE-Cadherin). For example, at least about 70%,about at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 99% of the PVE-HE cells in apopulation may be CD105+, CD144+, and CD31+. In certain embodiments thePVE-HE cells are loosely adherent to each other. CD31, the plateletendothelial cell adhesion molecule-1 (PECAM-1), has been used as amarker for the development of endothelial cell progenitors,vasculogenesis, and angiogenesis. CD31 is constitutively expressed onthe surface of adult and embryonic endothelial cells, is a majorconstituent of the endothelial cell intercellular junction (where up to10⁶ PECAM-1 molecules are concentrated) and is weakly expressed on manyperipheral leukocytes and platelets.

In exemplary embodiments, PVE-HE may exhibit one or more, preferablyall, of the following characteristics: (1) Significant amount ofCD31+PVE-HE cells can be detected as early as 72 hours after initiationof PVE-HE differentiation. (2) It will reach peak level at round 120 to146 hours after initiation of PVE-HE differentiation. (3) TheCD31+PVE-HE cell population can be isolated and cryopreserved. (3) Theyexpress almost all endothelial progenitor cell surface marker such asCD31, CD105 (Endoglin), CD144 (VE-Cadherin). They may also express CD34,CD309 (KDR) and CD146. (4) A subpopulation of CD31+PVE-HE cells alsoexpress CXCR4 (CD184). (5) The endothelial lineage potential can beconfirmed by culturing CD31+PVE-HE cells onto fibronectin in endothelialcell (EC)-specific medium (such as EGM-2 or EndoGro) to obtain monolayerof cells with typical endothelial morphology. (6) PVE-HE-derivedendothelial cells (PVE-HE-EC) not only express CD31 (localized atcell-cell junction), but also express von Willibrandt Factor (vWF), andcapable of LDL-uptake. (7) PVE-HE-ECs are capable of forming 3D-networkstructure when cultured on top of Matrigel. (8) When plating in extremelow density on Fibronectin in EC-specific medium, the CD31+PVE-HE cellsare capable of forming colonies with typical endothelial morphologyconfirming their clonogenic capability. (9) When plating inmethylcellulose medium for blast-colony growth, blast colony can only begenerated from CD31+ fraction. Unlike CD31− cells, both CD34− and CD105−are capable of generating blast colonies suggesting hemogenic capabilityis exclusively maintained in CD31 fraction only. (10) Newly derivedPVE-HE-EC maintain hemogenic potential and will give rise to hemogeniccells if cultured under conditions favorable to the hematopoieticlineages.

The term “megakaryocyte lineage-specific progenitor cells” (“MLPs”), asused herein, refers to mononuclear hematopoietic stem cells committed toat least the megakaryocyte lineage and includes, but is not limited to,cells in the umbilical cord blood, bone marrow, and peripheral blood aswell as hematopoietic stem cells, cells derived from human embryonicstem cells, and cells derived from induced pluripotent stem cells. Thesecells can be described based on numerous structural and functionalproperties including, but not limited to, expression (RNA or protein) orlack of expression (RNA or protein) of one or more markers. The MLPs ofthis disclosure can be a mixture of immature and mature cells. Thepercentage of mature vs. immature MLPs may vary based upon the length oftime in culture in MLP-Derivation and Expansion Medium (MLP-DEM, alsoreferred to herein as APEL) (described in Example 2). Immature MLPs arecharacterized by the expression of the markers CD41a, CD31, CD34, CD13and the lack of CD14 and CD42b marker expression. Exemplary methods ofthe disclosure provide for detection and/or purification of MLPs by amethod comprising detecting the expression of CD13 and/or enriching orpurifying CD13-positive cells. Optionally in these methods theexpression of CD13 may be detected and/or used as a basis for cellpurification in combination with the expression of one or moreadditional markers of immature or mature MLPs described herein. MatureMLPs are characterized by the expression of the markers CD41a, CD31,CD34, CD13, CD42b and the lack of CD14 expression. In certainembodiments MLPs generated in feeder free culture may be semi-detachedor detach completely and may float in the culture medium. For example,MLPs may be collected from the PVE-HE when they start to float up insuspension. Preferably MLPs are not plated and allowed to adhere, whichmay permit differentiation into lineages other than MKs or non-MKlineages (such as endothelial cells). MLPs are preferably grown insuspension to produce MKs. Optionally, MLPs may be cryopreserved.

The term “megakaryocytes” (MKs) as used herein refers to large polyploidhematopoietic cells that give rise to platelets, as well as smaller MKs(which may be produced by the subject methods) that may be diploid butfully capable of producing platelets. One primary morphologicalcharacteristic of mature MKs is the development of a large, polyploidnucleus. Mature MKs stop proliferating but continue to increase theirDNA content through endomitosis; with a parallel increase in cell size.The large polyploid nucleus, large cell volume, and ample cytoplasm ofMKs allows for the production of thousands of platelets per cell. MKscan be described based on these and numerous other structural andfunctional properties including, but not limited to, expression (RNA orprotein) or lack of expression (RNA or protein) of one or more markers.Mature MKs express the markers CD41a and CD42b. Mature MKs may alsoexpress CD61 and CD29. For example, a MK of the present disclosure ispreferably functional in the sense of being able to produce platelets,e.g., when cultured under conditions those described herein. Exemplaryembodiments provide a method of detecting matured MKs comprisingdetecting expression of CD29 and identifying CD29 positive cells asmatured MKs. Additional exemplary embodiments provide a method ofpurifying MKs, comprising purification of CD29 positive cells from apopulation, for example using magnetic bead subtraction, FACS, otherimmunoaffinity based methods, or the like. Optionally in these methodsthe expression of CD29 may be detected and/or used as a basis for cellpurification in combination with the expression of one or moreadditional markers of matured MKs, such as those markers shown in Table1 (which provides further exemplary cell surface marker expression ofmatured MKs).

In vivo, MK are derived from hematopoietic stem cell precursor cells inthe bone marrow. These multipotent stems cells reside in the marrowsinusoids and are capable of producing all types of blood cellsdepending on the signals they receive. The primary signal for MKproduction is TPO. TPO induces differentiation of progenitor cells inthe bone marrow towards a final MK phenotype. The MK develops throughthe following lineage: CFU-ME (pluripotent hemopoietic stem cell orhemocytoblast), megakaryoblast, promegakaryocyte, megakaryocyte. Thecell eventually reaches megakaryoblast stage and loses its ability todivide. However, it is still able to replicate its DNA and continuedevelopment, becoming polyploidy. The cytoplasm continues to expand andthe DNA complement can increase to greater than 64 N.

Once the cell has completed differentiation and becomes a maturemegakaryocyte, it begins the process of producing platelets. TPO plays arole in inducing the MK to form small proplatelet processes. Plateletsare held within these internal membranes within the cytoplasm of the MK.There are two proposed mechanisms for platelet release. In one scenario,these proplatelet processes break up explosively to become platelets.Alternatively, the cell may form platelet ribbons into blood vessels.The ribbons are formed via pseudopodia and they are able to continuouslyemit platelets into circulation. In either scenario, each of theseproplatelet processes can give rise to 2000-5000 new platelets uponbreakup. Overall, more than 75% of these newly-produced platelets willremain in circulation while the remainder will be sequestered by thespleen.

The term “platelet” as used herein refers to anucleate cytoplasmicbodies derived from cells that are involved in the cellular mechanismsof primary hemostasis leading to the formation of blood clots. Platelets(thrombocytes) are small, irregularly shaped clear cell fragments 2-3 pmin diameter, which in vivo are derived from fragmentation of precursormegakaryocytes (MK). Platelets can be identified based on numerousstructural and functional properties including, but not limited to,expression (RNA or protein) or lack of expression (RNA or protein) ofone or more markers. Platelets express the markers CD41a and CD42b.Platelets adhere to tissue and to each other in response to vascularinjury.

The terms “functional platelet” or “platelets that are functional” asused herein refer to platelets that can be activated by thrombin andparticipate in clot retraction. In exemplary embodiments, whetherplatelets are functional platelets may be determined using an animalmodel, e.g., as described in Example 4 (FIG. 12), for example bycomparison to naturally-derived platelets, which may be of the samespecies. Additionally, activated platelets can be identified byexpression of CD62p and αIIbβIII, and functional platelets can beidentified (optionally quantitatively) by their expression of thesemarkers upon activation such as upon activation with thrombin. Thephrase “substantially all of the platelets are functional” refers to acomposition or preparation comprising platelets wherein, for example, atleast 60% of the platelets are functional platelets, or at least 65%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% of the platelets are functionalplatelets. Functional platelets may be active for at least 5 days whenstored at 22-37° C.

PECAM1 (CD31) is a member of the immunoglobulin (Ig) superfamily that isexpressed on the surface of circulating platelets, monocytes,neutrophils, and particular T-cell subsets. It is also a majorconstituent of the endothelial cell intercellular junction, where up toan estimated 1 million molecules are concentrated. Because of thiscellular expression pattern, PECAM1 is implicated in several functions,including transendothelial migration of leukocytes, angiogenesis, andintegrin activation. Ig superfamily mediate cell adhesion (e.g., NCAM1,ICAM1, and VCAM1) or antigen recognition (e.g., immunoglobulins, T-cellreceptors, and MHC molecules). In addition, a subgroup comprising 30members characterized by the presence of 1 or more immunoreceptortyrosine-based inhibitory motifs (ITIMs) within their cytoplasmicdomains has also been recognized. PECAM1, which has 6 ITIMs within itscytoplasmic domain, is a member of this subfamily.

Endoglin (ENG), also called CD105, is a homodimeric membraneglycoprotein primarily associated with human vascular endothelium. It isalso found on bone marrow proerythroblasts, activated monocytes,fibroblasts, smooth muscle cells and lymphoblasts in childhood leukemia.Endoglin is a component of the transforming growth factor-beta (TGFB)receptor complex and binds TGFB1 with high affinity. Endoglin isinvolved in the cytoskeletal organization affecting cell morphology andmigration, in processes such as development of cardiovascular system andin vascular remodeling. Its expression is regulated during heartdevelopment. Experimental mice without the endoglin gene die due tocardiovascular abnormalities.

VE-cadherin (CD144) is a classical cadherin from the cadherinsuperfamily. VE-cadherin plays an important role in endothelial cellbiology through control of the cohesion and organization of theintercellular junctions, therefore maintain the integrity of theendothelium. VE-cadherin is indispensable for proper vasculardevelopment. Transgenic mouse models studies confirmed that VE-cadherindeficiency is embryonically lethal due to vascular defects. VE-cadherinserves the purpose of maintaining newly formed vessels.

The term “ROCK inhibitor” as used herein refers to any substance thatinhibits or reduces the function of Rho-associated kinase or itssignaling pathway in a cell, such as a small molecule, an siRNA, amiRNA, an antisense RNA, or the like. “ROCK signaling pathway,” as usedherein, may include any signal processors involved in the ROCK-relatedsignaling pathway, such as the Rho-ROCK-Myosin II signaling pathway, itsupstream signaling pathway, or its downstream signaling pathway in acell. An exemplary ROCK inhibitor that may be used is Stemgent'sStemolecule Y27632, a rho-associated protein kinase (ROCK) inhibitor(see Watanabe et al., Nat Biotechnol. 2007 June; 25(6):681-6) Other ROCKinhibitors include, e.g., H-1152, Y-30141, Wf-536, HA-1077,hydroxyl-HA-1077, GSK269962A and SB-772077-B. Doe et al., J. Pharmacol.Exp. Ther., 32:89-98, 2007; Ishizaki, et al., Mol. Pharmacol.,57:976-983, 2000; Nakajima et al., Cancer Chemother. Pharmacol.,52:319-324, 2003; and Sasaki et al., Pharmacol. Ther., 93:225-232, 2002,each of which is incorporated herein by reference as if set forth in itsentirety. ROCK inhibitors may be utilized with concentrations and/orculture conditions as known in the art, for example as described in USPGPub No. 2012/0276063 which is hereby incorporated by reference in itsentirety. For example, the ROCK inhibitor may have a concentration ofabout 0.05 to about 50 microM, for example, at least or about 0.05, 0.1,0.2, 0.5, 0.8, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45,or 50 microM, including any range derivable therein, or anyconcentration effective for promoting cell growth or survival.

For example, pluripotent stem cell viability may be improved byinclusion of a ROCK inhibitor. In an exemplary embodiment, thepluripotent stem cells may be maintained under feeder-free conditions.In another example, megakaryocyte lineage-specific progenitor cellviability may be improved by inclusion of a ROCK inhibitor. In anotherexample, megakaryocyte viability may be improved by inclusion of a ROCKinhibitor. In another exemplary embodiment, the megakaryocytelineage-specific progenitor cell may be maintained under feeder freeconditions.

Abbreviations

iPS: induced pluripotent stem

iPSC: induced pluripotent stem cell

hiPSC: human induced pluripotent stem cell

hES: human embryonic stem

hESC: human embryonic stem cell

MK: megakaryocyte

MLP: megakaryocytic lineage-specific progenitor also calledmegakaryocyte progenitor (MKP)

PVE-HE: hemogenic endothelial cell, which are optionallyPECAM1+VE-Cadherin+Endoglin+ and which are optionally derived frompluripotent stem cells,

iPS-PVE-HE: iPS cell-derived hemogenic endothelial cell (such as aPECAM1+VE-Cadherin+Endoglin+ cell)

hES-PVE-HE: hES cell-derived hemogenic endothelial cell (such as aPECAM1+VE-Cadherin+Endoglin+ cell)

PVE-HE-MLP: megakaryocytic lineage-specific progenitor produced from ahemogenic endothelial cell

iPS-PVE-HE-MLP: PVE-HE-MLP produced from an iPS cell

hES-PVE-HE-MLP: PVE-HE-MLP produced from an hES cell

PVE-HE-MLP-MK: megakaryocyte produced from a megakaryocyticlineage-specific progenitor which was produced from a hemogenicendothelial cell

iPS-PVE-HE-MLP-MK: PVE-HE-MLP-MK produced from an iPS cell

hES-PVE-HE-MLP-MK: PVE-HE-MLP-MK produced from an hES cell

PLT: platelet

hiPSC-PLT or iPSC-platelet: platelet or platelet-like particle producedfrom human induced pluripotent stem cells

hESC-PLT: platelet or platelet-like particle produced from humanembryonic stem cells

ADM: advanced differentiation morphology.

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All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Generation of Pluripotent-Derived Hemogenic Endothelial Cells(PVE-HE)

Pluripotent-Derived Hemogenic Endothelial Cells (PVE-HE) were generatedfrom induced pluripotent stem (iPS) cells.

First, iPS cells were expanded by culturing on Matrigel (a solublepreparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) in thefeeder-free pluripotent stem cell culture medium mTeSR1. Briefly, humaniPS cells were harvested by dissociation using chemically defined CellDissociate Buffer (CDB) with EDTA as the sole active component. Noenzyme or any other animal products were used in the culture medium orCDB. For investigators with ordinary skill in the art, it should beunderstood that it is possible to use another chemically defined matrixsuch as recombinant vitronectin or SyntheMax II, or medium such asmTeSR2, or other compatible cell dissociation reagents.

Second, harvested human iPS cells were prepared for differentiation intomultipotent PVE-HE which were PECAM1+VE-Cadherin+Endoglin+. Embryoidbody (EB) formation was not required. Briefly, the harvested cells wereresuspended in mTeSR1, and plated on top of the extracellular matrixhuman collagen IV (Advanced BioMatrix, cat #5022). The small moleculeROCK inhibitor Y27632 was added to the culture at 10 μM and was thoughtto help the harvested iPS cells attach to the Collagen IV coatedsurface. The iPS cells were allowed to attach for 12-48 hours in at 37°C. with 5% CO2. As shown in FIG. 2A, after 48 hours attached cells showtypical pluripotent stem cell morphology under feeder-free condition.

Third, prepared human iPS cells were differentiated into PVE-HE cells.Briefly, mTeSR1 medium with Y27632 was removed and a differentiationinitiation medium (DIM) was added. DIM is an animal-component freemedium (ACF) comprised of Iscove's Modified Dulbecco's Medium (IMDM) asbasal medium, human serum albumin, iron-saturated transferrin, insulin,b-mercaptoethanol, soluble low-density lipoprotein (LDL), cholesterol,bone morphogenetic protein 4 (BMP4) at 50 ng/ml, basic fibroblast growthfactor (bFGF) at 50 ng/ml, and vascular endothelial growth factor (VEGF)at 50 ng/ml. After being incubated for 48 hours at 37° C. with 5% CO₂,early morphology of desired PVE-HE differentiation was observed. Thealmost complete transition from pluripotent stem cell morphology intoscattered small cell clusters can be seen in FIG. 2B. After 96 to 146hours post PVE-HE differentiation initiation, advanced differentiationmorphology was observed showing small compact cell clusters growing ontop of the monolayer (FIG. 2C).

Fourth, after 120 hours post PVE-HE differentiation initiation, advanceddifferentiation morphology (ADM) cells were analyzed for morphologicchanges (FIG. 3B) and successful PVE-HE differentiation. Briefly, asmall sample of cells was subjected flow cytometric analysis oflineage-specific markers CD31 (PECAM), CD105 (Endoglin), CD144(VE-Cadherin). The ADM cells show the PVE-HE phenotype CD31⁺CD144⁺CD105⁺at this stage of differentiation (FIG. 3A).

Example 2 Generating Detached Megakaryocytic Lineage-SpecificProgenitors (MLPs) from Human iPS-Derived PVE-HE Cells

First, initiation of MLP differentiation was performed 120 hours afterinitiating PVE-HE differentiation. Briefly, DIM medium containing 50ng/ml BMP4, 50 ng/ml bFGF, and 50 ng/ml VEGF was removed and replacedwith MLP Derivation and Expansion medium (MLP-DEM, also referred to asAPEL or Stemline II, as shown in FIG. 1) comprised of Iscove's modifiedDulbecco's medium (IMDM), Ham's F-12 nutrient mixture, Albucult (rhAlbumin), Polyvinylalcohol (PVA), Linoleic acid, SyntheChol (syntheticcholesterol), Monothioglycerol (a-MTG), rhInsulin-transferrin-selenium-ethanolamine solution, protein-freehybridoma mixture II (PFHMII), ascorbic acid 2 phosphate, Glutamax I(L-alanyl-L-glutamine), Penicillin/streptomycin, Stem Cell Factor (SCF)at 25 ng/ml, Thrombopoietin (TPO) at 25 ng/ml, Fms-related tyrosinekinase 3 ligand (FL) at 25 ng/ml, Interleukin-3 (IL-3) at 10 ng/ml,Interleukin-6 (IL-6) at 10 ng/ml, and Heparin at 5 Units/ml. Cells werethen incubated for up to 8 days at 37° C. with 5% CO₂. MLPdifferentiation was maintained for 8 days.

TABLE 1 MLP/MK marker comparison Matured MLPs Matured MKs CD43 95.9%98.5% CD41 89.3% 91.4% CD61 77.2% 89.7% CD42a 66.3% 88.8% CD144 57.6%15.1% CD31 57.0% 80.6% CD29 40.4% 96.2% CD45 40.3% 66.1% CD13 40.4%59.4% CD34 30.3% 44.1% CD309 28.4% 73.2% CD71 26.4% 59.6% CD90 19.8% 2.0% CD105  7.5% 46.0% CD56  3.5%  3.3% CD14  4.4%  4.7% CD143  2.2% 6.8% CD15  1.6% 11.0% CD3  2.8%  4.2% CD117  2.7% 10.2% CD184  0.6% 8.5% CD11c  0.4%  2.5% CD73  0.0%  1.5%

Table 1 shows comparative flow cytometric analysis of marker expressionby matured MLP and matured MK cells. The analysis characterizes thephenotype of PVE-HE-derived MLP cells prior to crypopreservation andsubsequent differentiation into MK cells of the defined phenotype.

Second, floating and semi-detached MLPs on top of the attached cellpopulation were collected by wash with mild force using serologicalpipette. Medium containing these MLPs was transferred into conical tubesand centrifuge at 300×g for 5 minutes to collect the MLPs. Medium wasdiscarded and MLP pellet was resuspended in phosphate buffered salineand analyzed for morphology (FIG. 5) and by flow cytometry using markersseen in Table 1. Selected results seen in FIG. 4. Results demonstratethat MLPs collected at this stage are mainly (over 90%) comprised of twopopulations, one characterized by a relatively immature MLP populationrepresented by CD41a⁺CD31⁺CD34⁺CD14⁻CD13⁺CD42b⁻, and a more matured MLPspopulation represented by CD41a⁺CD31⁺CD34⁺CD14⁻CD13⁺CD42b⁺.

Third, MLP cells were cryopreserved. Briefly, cryopreservation of MLPswas achieved using the cell freezing medium CS10 (Sigma) containing 10%DMSO.

Example 3 Generating Mature Platelets from Human iPS-PVE-HE-MLP-DerivedMegakaryocytes (MK)

First, initiation of platelet differentiation was performed using humaniPS-PVE-HE-derived MLP cells, as described above. MLPs were seeded ontoa non-adherent surface in MK media (MK-M) comprised of Iscove's ModifiedDulbecco's Medium (IMDM) as basal medium, human serum albumin,iron-saturated transferrin, insulin, b-mercaptoethanol, solublelow-density lipoprotein (LDL), cholesterol, TPO at 30 ng/ml, SCF at 1ng/ml, IL-6 at 7.5 ng/ml, IL-9 at 13.5 ng/ml, Y27632 at 5 μM, andHeparin at 5-25 units/ml. Cells were then incubated at 37° C. in 5% CO₂for 3 days.

In some instances, the MLPs were seeded onto a non-adherent surface inculture medium comprised of Iscove's modified Dulbecco's medium (IMDM),Ham's F-12 nutrient mixture, Albucult (rh Albumin), Polyvinylalcohol(PVA), Linoleic acid, Linolenic acid, SyntheChol (syntheticcholesterol), Monothioglycerol (a-MTG), rhInsulin-transferrin-selenium-ethanolamine solution, protein-freehybridoma mixture II (PFHMII), ascorbic acid 2 phosphate, Glutamax I(L-alanyl-L-glutamine), Penicillin/streptomycin, TPO at 30 ng/ml, SCF at1 ng/ml, IL-6 at 7.5 ng/ml, IL-9 at 13.5 ng/ml, Y27632 at 5 μM, andHeparin at 5-25 units/ml. Cells were then incubated at 37° C. in 5% CO₂for 3 days.

Second, iPS-PVE-HE-MLP-derived maturing MK cells were analyzed. At 72hours post initiation of platelet differentiation, very large polyploidMKs (50 μM) became abundant with progression of maturation (FIGS. 5A &B. Scale bar in 5A is 100 μM, N in 5B indicates nucleus inside MK). Frombetween 72-96 hours proplatelet forming cells with elongated pseudopodiawere readily observed microscopically. (FIGS. 5A & 5C, indicated byarrows), and emerging amounts of CD41a+CD42b+ platelet particles weredetected using flow cytometric analysis (FIG. 6). In FIG. 6 the cells in“A” were circulating human platelets, “B” is hES-PVE-HE-MLP, “C” isperipheral blood derived human platelets, “D” is iPS-PVE-HE-MLP, and “E”is hES-PVE-HE-MLP). At 84 hours post initiation, the amount ofCD41a+CD42b+ platelets increased dramatically, reaching levels as highas 70% (FIG. 6D).

Third, high quality platelets were harvested without harming maturingMKs in cultures for at least 3-5 consecutive days and possibly longer,maximizing the yield of platelets from MLPs at least 3-5 times. Toseparate large MKs from platelet containing media, the cell suspensionwas centrifuged at 50×g for 10 minutes, supernatant removed and MK cellpellets re-suspended with fresh MK-M, allowing them to produce moreplatelets later. To separate platelets from proplatelets, large celldebris and small MKs, a BSA/HSA gradient sedimentation method wasapplied to obtain a purer population of platelets from the supernatantof the 50×g centrifugation. Purified platelets were suspended with MK-Mand maintained at room temperature. Quality of platelets with respect togranularity, transparency, size, and cell surface marker expression werecharacterized by FACS analysis and compared to peripheral blood derivedhuman platelets. (FIG. 6A-E). Purified platelets were stored in MK-Mmedia at room temperature within a non-adherent surface, securing aminimal loss of functional platelets.

Example 4 Analysis of Mature Platelets from iPS-PVE-HE-MLP-DerivedMegakaryocytes (MK)

First, iPS-PVE-HE-MLP-derived platelets (hiPSC-PLTs) were analyzed formorphology. Immunoflouresensce and transmission electron microscopicanalysis was performed according to methods previously described (CellResearch 2011 21:530-45). hiPSC-PLTs were found to be discoid and mostlyultrastructurally identical to circulating human PLTs (as demonstratedby transmission electron microscopy, FIG. 7). hiPSC-PLTs were comparableon average size with circulating human PLTs (2.38 μm±0.85 μm versus 2.27μm±0.49 μm) as demonstrated by DIC and β1-tubulin IF microscopy (FIG.8). hiPSC-PLTs spread on glass—form both filopodia and lamelopodia (asdemonstrated by DIC live-cell microscopy images shown in FIG. 9).hiPSC-PLTs were anucleate, and comparable to circulating human PLTs (asdemonstrated by Hoechst staining, FIG. 8). hiPSC-PLTs were seen to havenormal tubulin cytoskeleton relative to circulating human PLTs (asdemonstrated by β1-tubulin labeling, FIG. 8). hiPSC-PLTs were seen tohave normal filamentous actin relative to circulating human PLTs (asdemonstrated by phalloidin labeling, FIG. 8). hiPSC-PLTs were seen tohave normal alpha-granule expression relative to circulating human PLTs(as demonstrated by TSP4 and PF4 labeling in FIGS. 10A & 10B).

Second, hiPSC-PLTs were analyzed for functionality using an in vitroactivation assay measuring the cell adhesion molecule expression on anactivated platelet. Briefly, two adhesion molecules were analyzed, usingan anti-CD62p antibody, and the PAC-1 antibody. PAC-1 recognizes theαIIbβIII integrin. Both CD62p and αIIbβIII are expressed on the surfaceof activated platelets. The assay was performed according to methodsdescribed previously (Cell Research 2011 21:530-45). In response tothrombin exposure, both PAC-1 and P-selectin binding were increased(FIG. 11).

Third, hiPSC-PLTs were analyzed for functionality using an in vivo assaysystem measuring thrombus formation in macrophage-depleted NOD/SCIDmice. Briefly, intravital microscopy of cremaster muscle arterioles wasperformed as previously described (Cell Research 2011 21:530-45).Macrophage-depleted NOD/SCID mice were anesthetized by intraperitonealinjection of ketamine (125 mg/kg) and xylazine (25 mg/kg). A trachealtube was inserted and the mouse was placed on a thermo-controlledblanket. After incision of the scrotum, the cremaster muscle wasexteriorized onto an intravital microscopy tray. The muscle preparationwas superfused with thermo-controlled (37° C.) and aerated (95% N2, 5%CO2) bicarbonate-buffered saline throughout the experiment. Thecremaster muscle arteriolar wall was injured by micropoint laserablation using a Micropoint Laser System (Photonics Instruments). Thedeveloping mouse platelet thrombus was visualized by infusion of Dylight649-conjugated anti-mouse CD42c antibodies (Emfret Analytics, 0.05 μg/gbody weight) through a jugular cannulus. Calcein AM-labeled hPLT,iPSC-PLT, and ESC-PLT, 3×10⁶, were also infused with or without ReoPro,100×g, into mice. Two to four thrombi were generated in one mouse.Fluorescence and brightfield images were recorded using an Olympus BX61Wmicroscope with a 60 x/1.0 NA water immersion objective and a high speedcamera (Hamamatsu C9300) through an intensifier (Video ScopeInternational). Data were collected for 5 min following vessel wallinjury and analyzed using Slidebook v5.0 (Intelligent ImagingInnovations). The results seen in FIG. 12A are an indication thathiPSC-PLTs contribute to clot formation in an in vivo setting.hiPSC-PLT's functional capabilities were also determined to be mediatedby αIIbβIII. Specifically, hiPSC-PLTs were pretreated with ReoPro, a Fabfragment of a human-murine chimeric monoclonal antibody that bindsspecifically to αIIbβIII and inhibits platelet function (FIG. 12B).Asterisks indicate a statistically significant difference compared tocontrols not treated with ReoPro (p<0.01 versus control, Student'st-test).

Fourth, the kinetics of hiPSC-PLTs in macrophage-depleted NOD/SCID miceafter infusion were determined. To determine whether the kinetics ofhiPSC-PLT and ESC-PLT is similar to that of hPLT in vivo, iPSC- orESC-PLTs were infused into macrophage-depleted NOD/SCID mice, and bloodcollected at various time points was analyzed by flow cytometry.Briefly, macrophages were depleted by intravenous injection ofliposome-encapsulated clodronate as described previously.Clodronate-liposomes were injected into mice through a tail vein at Day0 (100 μl) and Day 2 (50 μl). At Day 3, human platelet-rich plasma wasobtained by centrifugation of sodium citrate-treated blood at 200×g for25 min and centrifuged at 800×g for 10 min in the presence of 0.5 μMPGE1 and 10% citrate buffer. The pellet was resuspended withHEPES-Tyrode buffer containing 0.15 μM PGE1 and 10% citrate buffer andcentrifuged at 800×g for 5 min. Platelets were resuspended inHEPES-Tyrode buffer containing 0.1% fatty acid-free BSA. Isolated humanplatelets (hPLT, 1.5×10⁷), induced pluripotent stem cell-derivedplatelets (hiPSC-PLT, 1.5×10⁷) of the present disclosure, and embryonicstem cell-derived platelets (ESC-PLT, 1.0×10⁷) of the present disclosurewere intravenously infused into macrophage-depleted mice. Mouse blood,30 μl, was collected through a jugular vein at different time points(10, 30, 60, 120, 240, 360, and 480 minutes) and analyzed by flowcytometry using APC-conjugated anti-human CD41 and Dylight488-conjugated anti-mouse CD42c antibodies. Results demonstrate thathiPSC-PLTs circulate for several hours in macrophage-depleted NOD/SCIDmice (FIG. 13).

Example 5 Production of Platelets from Pluripotent Cells

This example provides further exemplary methods of producing plateletsfrom pluripotent stem cells.

As noted above, hESC-PLT and hiPSC-PLT are human platelets which areproduced ex vivo from human embryonic and induced pluripotent stemcells. Initial in vitro and in vivo characterization described above hasdemonstrated that both hESC-PLT and hiPSC-PLT are morphologically andfunctionally comparable to human donor platelets. For example, hiPSC-PLTwere able to spread on a glass substrate.

The production of bulk intermediate (MK progenitors) is initiated withthe thaw of vials from an approved hiPSC or hESC master cell bank (MCB).The process flow chart for the production of bulk intermediate ispresented in FIG. 14A-E. Cryovials containing 1-2 million hiPSC or hESCmaster or working cell banks are removed from the vapor phase of cGMPliquid nitrogen storage and transferred to the clean room (ISO Class 7).All processing is performed in a certified biosafety cabinet (ISO Class5 BSC). Following thaw and wash to remove DMSO; cells are seeded onMatrigel coated vessels in mTeSR1 defined culture media. Care is takento maintain the cells as aggregates. Plates are labeled with the date,lot number, and passage number and the lid of each well is labeled aunique number (e.g. 1-6). Seeded plates are placed in a 37° C., 5% CO₂incubator. Cultures are inspected daily using a stereo-microscope and aninverted light microscope. Observations regarding colony size, cellularmorphology; including the extent of differentiation, and media color arerecorded on a work sheet. mTeSR1 medium is changed typically every 1-2days until cultures are 60-90% confluent. When morphology and confluenceevaluations indicate that the cultures need to be passaged, colonies areharvested using cell dissociation buffer. Care is again taken tomaintain the cells as aggregates. Cultures are re-seeded onto Matrigelcoated vessels in mTeSR1 media. Based on the cm² harvested and the cm²to be seeded, cells are typically split at a ratio of 1:4 to 1:8 every3-7 days depending on the yield of stem cells required. Seeded culturesare returned to a 37° C., 5% CO₂ incubator. Stem cells may be passed 1-6times before induction of hemogenic differentiation depending on therequired lot size.

Three day post-seeding for hemogenic cell differentiation, cultures areexamined to confirm cell attachment and outgrowth. At this stagecultures are typically low density with attached cells covering lessthan 10% of the total surface area. Well attached cells shoulddemonstrate outgrowth with a significant transition from pluripotentstem cell morphology to differentiated cells: larger cells withsignificantly more cytoplasm relative to the size of the nucleus growingmore diffusely with no clear colony borders.

When the stem cell expansion phase is estimated to meet cell yieldrequirements, hemogenic differentiation is initiated. A representativevessel is sacrificed for harvest and a single cell suspension iscreated. The cell concentration is quantified to establish cultureseeding parameters, with the remaining cells discarded. Colonies arecarefully harvested using cell dissociation buffer and reseeded intocollagen IV coated vessels at a density of 5,000 cells/cm² in mTeSR1media supplemented with 10 uM Y27632 (ROCK inhibitor). Seeded vesselsare placed in a 37° C., 5% CO₂ incubator.

The following day the media is removed, taking care to minimize theremoval any floating cell clusters, and replaced with StemSpan ACF(StemCell Technologies Inc.) supplemented with recombinant human (rh)BMP-4 at 50 ng/ml, rh VEGF at 50 ng/ml, and rh bFGF at 50 ng/ml. Thecultures are transferred to a low O₂ (˜5%), 37° C., 5% CO₂ environmentand are left undisturbed for 2 days. After 2 days the cultures areevaluated morphologically for cell attachment and outgrowth. The mediais removed, taking care to minimize the removal any floating cellclusters, and replaced with fresh media. The cultures are returned tothe low O₂ (˜5%), 37° C., 5% CO₂ environment for an additional 2 days.Following this time period the media is changed once more and thecultures are placed in normoxic culture conditions. After 2 days ofnormoxic culture the culture media is removed, taking care to minimizethe removal of any floating cell clusters, and replaced with MLP mediumcomprising STEMdiff APEL (StemCell Technologies Inc.) supplemented with5 Units/ml heparin, rh 25 ng/ml TPO, 25 ng/ml rh SCF, 25/ng rh FL, rhIL-6 and rh IL-3. These conditions are maintained for the next 2-6 days.Cultures undergo daily morphological evaluation to assess the quality offloating MK progenitors. Media changes are not performed, but additionalmedia is added if the culture begins to indicate media depletion(culture media appears yellow). Cultures are periodically sampled andassayed by FACS for CD41a, CD42b expression. When culture morphology andCD41a, CD42b expression (˜≧15% double positive) are appropriate, thecultures are harvested Cells are then cryopreserved @ 3-5 million viableMLPs/mL/cryovial in 10% dimethyl sulfoxide, 90% fetal bovine serumcryopreservation medium (Hyclone). Cryopreservation is accomplished byplacing vials in freezing container (Nalgene) and then storing in a −80°C. freezer for 1-3 days, followed by transfer to the vapor phase of thecGMP liquid nitrogen storage system.

The production process for the final product hESC-PLT or hiPSC-PLT isinitiated with the thaw of approved bulk intermediate. Cryovialscontaining 3-5 million MLPs which have passed bulk intermediate qualitytesting are removed from the vapor phase of cGMP liquid nitrogen storageand transferred to the clean room. Cells are seeded into ultra-lowattachment (ULA) vessels at approximately 2.3×10⁵/cm² in StemSpan ACFmedia supplemented with 30 ng/ml rh TPO, 1 ng/ml rh SCF, 7.5 ng/ml rhIL-6, 13.5 ng/ml rh IL-9 and 5 uM Y27632 (ROCK inhibitor). Culturevessels are labeled with the date and lot number and the lid on eachvessel is labeled a unique number (e.g. 1-6). Cultures are maintained at39° C., in a humidified 10% CO₂ atmosphere. Typically there is nosignificant cell expansion during this culture phase. Approximately 3 to4 days into the culture, large maturing megakaryocytes (MK) will becomeevident. Cultures are monitored by periodic sampling and FACS analysisfor CD41a, CD42b expression. Proplatelet formation is typically firstobserved after five days in culture. As proplatelet formation progressesand CD41a, CD42b expression increases to approximately 30%-70% thecultures can be harvested (days 6-7).

The harvested media is centrifuged at 50×g to remove the megakaryocyteslurry. The supernatant is removed and centrifuged at 1000×g toconcentrate the platelets. The platelets are resuspended and then loadedon to a discontinuous albumin (Human) (HSA) gradient (12%, 10%, 7%, 5%and 2%) to further isolate the platelets. The platelet HSA gradient iscentrifuged at 80×g for 15 minutes. Platelets are harvested from thegradient and PGE₁ is added to the suspension to prevent activation.Platelets are concentrated by centrifugation at 1000 g for 10 minutes.Currently hESC and hiPSC platelets are stored in culture media. For thepurposes of the proposed study, a target product stability of 48 to 72hours will be investigated. Preliminary studies indicate a minimum of 24hour stability as determined by PAC1 binding results pre and poststorage.

FACS analyses (CD31 and CD43) of the MLP cell population in the bulkintermediate have shown that approximately 98% of the cells arecommitted to a hemogenic endothelial or hematopoietic lineage and thushave differentiated beyond pluripotency.

Further downstream in the process, cells present during MKdifferentiation and PLT harvest phase of manufacture have been tested byvWF (von Willebrand Factor) expression. Cells at this phase ofmanufacture are approximately 100% vWF+. The maintenance of a highlydifferentiated cell population indicates that the culture is notexperiencing a clonal expansion of undifferentiated progenitors.

Preliminary studies indicate a total absence of pluripotent cells inhiPS-derived MK cell populations analyzed by IFA staining for OCT4,NANOG, TRA-1-60, TRA-1-81 SSE3, SSE4 and Alkaline Phosphatase.Additional studies using IFA staining for pluripotent markers willexamine populations of hES-derived and hiPS-derived MLPs and MKs, aswell as, purified PLTs derived from the MKs, to screen for the presenceof pluripotent cells. Spiking studies will be conducted to determine theLODs of this assay to detect stem cells in the various cell populations.

Preliminary studies have been performed to characterize MK populationsusing FACs analyses for pluripotent markers (SSEA4 and TRA-1-60).Spiking studies have been performed where hES cells were mixed with MKsat 1% and 0.1% concentrations. The results indicate a limit of detectionof 0.1%. An initial study characterizing MKs for SSEA4 and TRA-1-60provided a result of <0.1% (below the assay level of detection)SSEA4/TRA-1-60 positive cells in the MK population.

Referring to FIG. 14, steps 2-3, since stem cells harvested withdissociation buffer are dislodged as cell clumps, accurate cell countsare not possible. For this reason, a representative culture vessel isharvested using Trypsin-EDTA to ensure a single cell suspension. Thissuspension is counted in a hemocytometer and the harvested cell numberis normalized per cm². Cells used for counting are then discarded. Theremaining product cultures are harvested using dissociation buffer andthe cell yield is calculated based on the normalized cells/cm²×cm²harvested. The calculated cell yield is used to set the required celldensity/cm² for seeding vessels to induce hemogenic celldifferentiation.

Referring to FIG. 14, step 10, beginning at 2 days in MLP medium and outto 6 days in MLP medium cell samples are taken from representativeculture vessels, pooled and assessed for the percentage of CD41a andCD42b doubly positive cells by FACS analysis. CD41a is a subunit offibrinogen receptor (αIIbβIII) and CD42b is a subunit on von WillebrandFactor receptor (GPIb-V-IX). The expression of both receptors isspecific for MK lineages and both are required for platelet function.Early lineage hemogenic endothelial cells are CD41a negative, expressingCD41a during late-stage hemogenic differentiation in hematopoieticprogenitors. CD42b is expressed exclusively in mature MKs. At thispoint, cultures are heterogeneous with a high percentage of CD41+ cellsand a low percentage of CD42+ cells (Pineault, et. Al., Megakaryocyteand platelet production from human cord blood stem cells. Methods MolBiol. 2012; 788:219-47).

The sample preparation and FACS analysis are performed as follows.Briefly, floating cells are collected and pooled. Using a serologicalpipette a gentle stream of growth medium is directed towards the culturesurface to detach any loosely adherent MLPs and pooled with thefree-floating cells. A sample (100-200 μL) of well-suspended pooledcells is collected and centrifuged (160×g for 5 minutes). The pellets isresuspended in DPBS, centrifuged again, and resuspended in DPBS plus 3%FBS (FACS buffer) containing CD41a-APC-conjugated (allophycocyanin) andCD42b-PE-conjugated (phycoerythrin) (BD Bioscience, San Jose, Calif.).Fluorescent conjugated antibodies and the appropriate isotype controls(mouse IgG1k-APC and mouse IgG1k-PE) are incubated in the presence for15 minutes at room temperature. Labeled cells are then diluted in FACSbuffer, centrifuged and resuspended in FACS buffer. FACS analysis isperformed by monitoring 10,000 events. Cultures with an acceptablepercentage of cells expressing double positive (CD41a+CD42b+) MLPs areharvested and cryopreserved. The tentative minimum specification is 10%double positive cells. Shown in FIG. 15 is a representative twodimensional dot plot for MLPs derived from hiPSC. In that Figure thecell population is 86.4% CD41a+; 31.2% CD42b+ with 29.9% of thepopulation staining double positive.

Prior to harvesting for cryopreservation, MLP cultures are assessed forthe approximate percentage of attached cells and the extent ofdifferentiated large cells with low nuclei to cytoplasm ratio. Attachedcells should appear as diffuse colonies with no clear colony borders.There should be an abundance of the floating MLPs resting on top of theattached cell population. Viable floating MLPs should appear clear, withminimal birefringence, demarked by a smooth cell membrane. Typically, asurface area of 1 cm² generates 5,000 to 15,000 MLPs per day. Shown inFIG. 16 is microphotograph of a representative population of MLPsderived from hiPSC line MA-iPS-01 (Hoffman Modulation Contrast, ×400)

Prior to cryopreservation MLPs, viable cell count is performed by trypanblue exclusion using a hemocytometer. Cells are assessed for the viablecell number and the percent viability. Representative vials are testedfor mycoplasma and sterility.

Additionally, a representative vial of cryopreserved MLPs is assessedfor CD31 and CD43 expression, as follows. CD31 is a marker for hemogenicendothelial cells expressed in both endothelial and hematopoieticlineages. Expression of CD43 confirms hematopoietic commitment. A samplevial of cryopreserved MLPs is thawed rinsed, resuspended in DPBS plus 3%FBS (FACS buffer) and centrifuged (160×g for 5 minutes). The pellet isresuspended in DPBS plus 3% FBS (FACS buffer) containingCD31-APC-conjugated (allophycocyanin) and CD43-FITC-conjugated (BDBioscience, San Jose, Calif.). Samples of thawed MLPs are incubated withthe fluorescent conjugated antibodies and the appropriate isotypecontrols (mouse IgG-APC and mouse IgG-FITC) for 15 minutes at roomtemperature. Labeled cells are then diluted in FACS buffer, centrifugedand resuspended in FACS buffer. FACS analysis is performed by monitoring10,000 events. Acceptable MLP banks have >/=50% CD31 positive cellsand >/=50% CD43 positive cells. Shown in FIG. 17 is a representative twodimensional dot plot for MLPs derived from hiPSC line MA-iPS-01. In theexample shown, the cell population is 99.8% CD31+; 98.5% CD43+ with98.4% of the population staining double positive.

Additionally, a representative vial of cryopreserved MLPs is assessed byFACS analysis utilizing pluripotent markers such as SSEA4, TRA-1-60 toconfirm the absence of pluripotent cells.

Sample cryovials of cryopreserved MLPs are thawed and assessed forviability and recovery post-thaw. MLPs are further processed to thepoint of proplatelet formation and assessed for morphology MK morphologyand FACS analysis for CD41a+ and CD42b+ during MK maturation. Culturesare monitored for proplatelet formation and for platelet formation andcharacterization by FACS analysis for doubly stained (CD41a+ and CD42b+)cells. Acceptance criteria include: sterility (negative on <USP 21>Immersion test), negative for mycoplasma (tested by Direct (agar &broth), Indirect (cell culture)), at least 90% positive for CD31 andCD43 by FACS, at least 70% viability by Trypan Blue, and negative forexpression of pluripotent cell markers.

Referring to FIG. 14, steps 13-14, after 2-3 days post-thaw and seedingof MLPs cultures are assessed for emergence of free-floating cells inthe MK lineage. At this time cells are heterogeneous in size rangingfrom 10-50 microns in diameter. The approximate proportion of viablecells is assessed by visual observation in an inverted light microscopewith healthy MK precursor cells and MKs displaying bright cytoplasm andsmooth cellular membranes. Shown in FIG. 18 is microphotograph of arepresentative population of MKs derived from hESC (Hoffman ModulationContrast, ×400).

Referring to FIG. 14, steps 13-14, after 2-3 days post-thawing of MLPs,cell samples are removed from representative culture vessels, pooled,processed, labeled with fluorescent-conjugated antibodies to CD41a andCD41b, and undergo FACS analysis. Cultures with an acceptable percentageof cells expressing both CD41a+ and CD42b+ MLPs are processed further(preferably at least 10% double positive cells).

Referring to FIG. 14, step 15, beginning at 3 days and out to 5 dayspost-thawing of MLPs, MK cultures are assessed for the appearance ofproplatelets. The arrows in FIG. 19 depict proplatelet morphology. MKdisplaying proplatelets exhibit long projections extending from thecells showing some beading and branching prior to fragmentation.

Referring to FIG. 14, steps 15-16, upon confirming the presence ofproplatelets, beginning at 3 days and out to 8 days post-thawing ofMLPs, samples of proplatelets and platelets are collected fromrepresentative cultures. Briefly, using a 10 mL serological pipette, thesamples are transferred to a 50 mL conical tube and drawn up andexpelled at least 5 times to generate shear force sufficient to fracturethe proplatelets. Samples are returned to a 39° C. incubator gassed with10% CO₂ and allowed to settle for 30 minutes. Approximately 250 μL ofthe supernatant containing suspended platelets is stained and undergoesFACS analysis for CD41a and CD42b. Platelet harvesting and subsequentprocessing is initiated when peak levels of platelets are detected,typically when 30-70% of the platelets are double positive stained(CD41a+CD42b+).

hiPSC-PLT and hESC-PLT are characterized for pH, platelet count andidentity (determined by expression of CD61, CD41a, CD42b), expression ofplatelet activation markers (CD62P, PAC1, Platelet Factor 4),Physiologic responses (Aggregation (microplate assay), TEG(thromboelastography), and Spreading), and morphologically evaluated byElectron Microcopy as well as DIC Microscopy with β1 Tubulin staining.

Cells are further tested for sterility (negative result of <USP 21>Immersion test), endotoxin (Gel Clot USP <85> less than 5 EU/ml),mycoplasma (negative result by European Pharmacopoeia and USPharmacopoeia test), identity (by FACS, at least 70% CD41a+, CD42b+),PLT Count/mpPLT (FACS for CD61), morphology (DIC Microscopy with β1Tubulin Staining), and potency by PAC1 Binding (Activated).

Purified platelets are stained with fluorescent-conjugated antibodies toCD41a and CD41b and undergo FACS analysis as described above. Shownbelow are representative two dimensional dot plot of PLTs derived fromiPS-01. In the example shown in FIG. 20, the population is 81.8% CD41a+;69.2% CD42b+ with 68.2% of the population staining double positive.

In conjunction with CD41a+, CD42b+ FACS performed in FIG. 20, propidiumiodine (PI) is added to the sample during CD staining to assessviability. FACS analysis is performed counting 10,000 events asdescribed previously, with additional events collected at flow channel 3to detect PI positive events (nonviable platelets). Data are analyzed togive the percentage of CD41a+/CD41b+ platelets and the percent viability(Total Platelets Detected−PI+Platelets/Total Platelets Detected).

Each final product lot of platelets is assessed for the number ofplatelets and the number of platelet microparticles by detecting CD61positive events using flow cytometric analysis. This assay is performedusing a known number of fluorescent beads to normalize data and obtainthe absolute number of PLTs/μL and mpPLTs/μL. Briefly, 5-10 μL samplesof platelets are diluted in 0.9% saline and dispensed into each of twotubes:

1) one TruCount (BD Cat #340334) tube containing a lyophilized pelletwith a known number of fluorescent beads and phycoerythrin-conjugatedanti-CD61 IgG in a final reaction volume of 60 μL/tube and

2) one isotype control tube containing PE-conjugated mouse IgG toaccount for nonspecific primary antibody binding in a final reactionvolume of 60 μL/tube.

The tubes are gently mixed, incubated for 20 minutes at 20-24° C.followed by the addition of 400 μL of cold (2-8° C.) 1% formaldehyde andstorage in the cold, protected from light for two hours. Prior toanalyzing the fixed platelet samples, the FACS machine settings for thepeak channel, gating, axes, quadrant locations, and marker boundariesfor the appropriate regions of data acquisition are determined bycollecting a minimum of 10,00 events from a sample of freshly sonicatedlatex beads with a uniform diameter of 1 μM (CML Cat #C37483) diluted in0.9% saline. After applying these settings, a minimum of 100,000 eventsis acquired for the isotype control and for the CD61 stained sample. Thecounts obtained on the CD61 sample will register events for CD-61positive fluorescence-stained PLTs, CD-61 positive mpPLTs and for thenumber of Trucount fluorescent beads detected. Events counted in thepreset regions are analyzed in the platelet size range 2-4 microns andin the mpPLTs quadrant corrected for nonspecific binding detected in theisotype control. Data are normalized to the efficiency of Trucount beadsdetected using the following formula based on the known number ofTrucount beads per tube provided by the manufacturer.

# Events Counted/# Beads Counted×# TruCount Beads per tube/SampleVolume=mpPLT/μL or PLT/μL.

Additionally, microscopic inspection is utilized to assess hESC-PLT andhiPSC-PLT morphology. Morphology is assessed by DifferentialInterference Contrast Microscopy (DIC) and by confirming staining forβ1-tubulin in a characteristic circumferential band of microtubulesunique to platelets. Briefly, platelets are fixed in 4% formaldehyde andcentrifuged onto 1 μg/ml poly-l-lysine-coated coverslips, permeabilizedwith 0.5% Triton X-100, and blocked in immunofluorescence blockingbuffer (0.5 g BSA, 0.25 ml of 10% sodium azide, and 5 ml FCS in 50 mlPBS) for a minimum of two hours before antibody labeling. To demarcatepermeabilized cells, samples are incubated with a rabbit polyclonalprimary antibody for human β1-tubulin generated against the C-terminalpeptide sequence CKAVLEEDEEVTEEAEMEPEDKGH (Genemed Synthesis, Inc.) (SEQID NO:1). Samples are then treated with a secondary goat anti-rabbitantibody conjugated to an Alexa Fluor 568 nm (Invitrogen; MolecularProbes), with extensive washes with PBS between and after theincubations. Coverslips are mounted (Aqua Polymount; Polysciences) ontomicroscope slides. As background controls, slides are incubated with thesecondary antibody alone and images are adjusted to account fornonspecific binding of antibodies. Samples are examined with amicroscope equipped with an oil immersion objective or differentialinterference contrast objective. Images are obtained using acharged-coupled device camera. Images were analyzed using the MetaMorphimage analysis software (Molecular Devices) and ImageJ (NationalInstitutes of Health). Characteristic features of resting platelets areassessed as follows: Differential-interference contrast (DIC)microscopy: Disc-shaped, approximately 2-3 μM in diameter β1-Tubulinstaining: A prominent, circumferential band of microtubules with adiameter similar to resting platelets. See FIG. 21.

Platelets (hESC-PLTs and hiPSC-PLTs) are additionally tested to confirmthe absence of pluripotent cells (e.g., by PCR, immunofluorescence,and/or FACS to detect pluripotency markers). To enhance sensitivity,detection methods may be coupled with procedures to concentratepotential cellular contaminants by trapping with filters, matrices orgradients coupled with low speed centrifugation to pellet cells andwhile leaving platelets in the supernatant.

Platelets (hESC-PLTs and hiPSC-PLTs) are additionally tested for thepresence of microorganisms according to the immersion method, USP <21>,CFR 610.12.

Platelets (hESC-PLTs and hiPSC-PLTs) are additionally tested forendotoxin. Gram-negative bacterial endotoxins are quantified using thePyrotell® Gel Clot Endotoxin System (Associates of Cape Cod, Inc.).Appropriate negative, positive, and positive product controls areprepared. Positive product controls are inhibition controls and consistof the specimen or dilution of specimen to which standard endotoxin isadded. The samples are added directly to the Pyrotell® reagent and mixedthoroughly. The reaction tubes are incubated at 37° C.±1° C. for 60±2minutes. A positive test is indicated by the formation of a gel whichdoes not collapse when the tube is inverted. Endotoxin is quantified byfinding the endpoint in a series of specimen dilutions. In the absenceof the endotoxin series, a positive control of know concentration may beincluded with the tests. The endotoxin assay will be validated for usewith PLT samples and is performed in a manner consistent with the 1987FDA guidance on endotoxin validation.

Platelets (hESC-PLTs and hiPSC-PLTs) are additionally tested formycoplasma. After centrifugation of platelets prior to running the HSAgradient, the supernatants (e.g., consisting of conditioned StemSpan ACFmedium) are collected and pooled. Samples of purified platelets from thefinal product batch and the conditioned medium are sent for mycoplasmatesting. Mycoplasma detection is performed as per the EuropeanPharmacopoeia and US Pharmacopoeia Guidelines with indirect cultivationon indicator cell cultures and direct inoculation on agar plates andinto broth.

Example 6 Assay for Platelet Potency and PAC-1 Binding

Platelets of the disclosure may be assessed for potency and/or PAC-1binding using the methods described in this example.

Platelet activation in vivo induces conformational changes in αIIbβ3integrin activating the fibrinogen receptor function of the GPIIb/IIIacomplex leading to enhanced ligand binding. Activated platelet bind tosubstrates including fibrinogen and Von Willebrand Factor and stimulatethrombus formation at the site of vascular injury. To determine theextent of functional αIIbβ3 integrin expression that occurs uponplatelet activation, hESC-PLTs, iPSC-PLTs, and purified normal humanplatelets are activated and assessed for the extent of PAC-1 binding ascompared to resting control PLTs. The PAC-1 is a fibrinogen mimetic thatbinds exclusively to the activated conformation of the αIIbβ3 integrin.

PLTs are counted and a minimum of 100,000 PLTs is removed and dilutedwith additional medium to a density of approximately 20 PLT/uL.PAC1-FITC (BD, 1:100 dilution) and antibodies (CD41a-APC-conjugated(allophycocyanin) 1:100 and CD42b-PE-conjugated (phycoerythrin) 1:100(BD Bioscience, San Jose, Calif.) are added to the PLT sample. One halfof the sample (250 uL) is dispensed into each of two 5 mL FACS tubes. Toone tube, thrombin (Sigma) is added to a final concentration of 1 U/mL.Activated PLTs exposed to thrombin and control samples are incubated atroom temperature for 15-20 minutes. Samples undergo FACS analysis withforward versus side scatter gating being determined using human bloodplatelets as controls. PAC-1 binding (activation) is quantified bycomparing the number of CD41a and PAC-1 positive events in the activatedto those in the unactivated control.

Example 7 Use of Proteases such as MMP Inhibitors to Improve Yields andPurity of Platelets Generated Under Shear Force

This disclosure also embraces platelet production methods that employshear force conditions (e.g., the culture medium is in flow during theculture. The culture was performed using a microfluidic device in whichflow rates in the range of tens of microliters/min approximate the shearforces in the bone marrow cavity during hemopoiesis. In some instances,the flow rate is in the range of 5-25 microliters/min. Megakaryocytesgenerated from iPS cells or ES cells according to this disclosure, whencultured under shear force, have been found to shed platelets in a muchmore efficient way.

The device used to culture the MLPs must be able to immobilize the MLPswithout attachment. In some instances, this means that the MLPs arelocated within a region or chamber of the device from which they areaccessible to the moving culture medium but not able to movesignificantly themselves.

This disclosure recognizes that platelet yield and purity can beimproved in these culture systems. In one aspect, the improvement isachieved through use of one or more certain protease inhibitors duringshear force culture. This Example demonstrates the benefit of adding amatrix metalloproteinase (MMP) inhibitor during these cultureconditions. It is to be understood that the MMP inhibitor isrepresentative of other protease inhibitors that similarly can be usedin these methods such as but not limited to plasminogen activatorinhibitors.

In another aspect, an improvement in platelet yield and purity isachieved by performing the culture at increased shear force. The shearforce may be 1, 1.5, 2, 2.5, 3, 3.5, 4 or 4.5 dynes/cm².

Matured MKs, derived from HA-iPS and MA09 hES lines, were used. Equalnumbers of MKs suspended in MK-specific medium were loaded into amicrofluidic device in the presence or absence of 20 μM MMP inhibitorGM6001. Constant shear force was applied to the MKs throughout theculture period. MK-medium downstream of MKs was collected at 30 minuteintervals for a period of 6 hours. Platelet number and purity (i.e.,percentage of total cells that are CD41a⁺CD42b⁺) in the collected mediawere measured using flow cytometer.

FIGS. 22-24 provide the results of platelet generation from MKs derivedfrom HA-iPS. The platelets were generated under a constant shear force.The addition of MMP inhibitor GM6001 at the beginning of the culturesignificantly improved the purity (FIG. 22) and yield (FIG. 24) of thenewly generated platelets. Purity is expressed at the percentage oftotal harvested cells that are CD41a⁺CD42b⁺. Increasing the viscosity ofthe culture medium by adding 8% dextran had a negative impact onplatelet formation.

FIG. 23 demonstrates that significantly more platelets were generatedfrom MKs under shear force culture when the MMP inhibitor GM6001, ispresent.

FIG. 24 demonstrates the difference in platelet yields in shear forcecultures in the presence of the MMP inhibitor GM6001 or DMSO, or under astatic culture.

FIG. 25 demonstrates that similar results are achieved when MA09 (ES)derived MKs were used to generate platelets. FIGS. 25 and 26 alsodemonstrate that a moderate change of shear force (represented by flowrate change from 12 to 16 μl/min) also improves platelet purity andyield.

Example 8 MMP8 Specific Inhibitor as a Protective Inhibitor Useful inPlatelet Formation in Static Culture

Proteases such as MMPs are involved in shedding of CD42b, which is alsocalled GPIbα. Platelet CD42b is the receptor for vWF and it mediatesinitial platelet involvement in wounds healing. Loss of CD42b cantherefore result in poor platelet quality. A broad-spectrum MMPinhibitor, GM6001, is reported to inhibit CD42b shedding from platelets.

In a comparative study using several specific MMP inhibitors, an MMP8inhibitor was identified as having more potency than GM6001 inprotecting platelet function. As shown in FIG. 26, the addition of anMMP8 specific inhibitor at the peak of platelet production of MKssignificantly improved the purity of iPS-derived platelets from 43.7% to65.5%, the latter of which is approximately 10% more than was obtainedfrom GM6001-treated cultures. Significantly more CD41a⁺CD42b⁺ plateletsare produced in the presence of MMP8 specific inhibitor as compared toGM6001. When the MMP8 specific inhibitor and GM6001 are used incombination, purity levels appear unaffected (FIG. 27) while plateletyields are increased (FIG. 28). The peak of platelet production isdetermined by measuring platelet content in the culture, for example ona daily basis for several days. It is typically 4-7 days after thebeginning of the MLP differentiation culture period, although it may belonger or it may be shifted.

While the data in FIGS. 27 and 28 were generated using iPS derivedmegakaryocytes, this disclosure provides for the use of an MMP8 specificinhibitor in cultures of naturally occurring sources of platelets suchas bone marrow and umbilical cord blood CD34⁺ progenitor cells.

Example 9 iPS Platelet Generation at Elevated Temperature

FIGS. 29 and 30 demonstrate that culturing megakaryocytes under mildhyperthermic conditions significantly improved both platelet purity andyield. The percentage of CD41a⁺CD42b⁺ platelets is significantly higherin cultures incubated at 39° C. than at 37° C. The effect is mostapparent prior to reaching the platelet production peak. In addition toimproving the purity, higher temperature incubation of megakaryocytesalso contributed to higher yield of platelet from the same startingnumber of megakaryocytes, indicating that elevated temperature has noadverse effect on megakaryocytes or platelets in the culture systemsprovided herein.

Example 10 iBET Promotes Megakaryocyte Commitment and Increases OverallPlatelet Yield Via Down-Regulation of c-myc Gene

In our new method of megakaryocyte lineage specific differentiation, theemergence of megakaryocyte progenitors best suited for plateletproduction can only be harvested in a short period of 3-4 days from thebeginning of the PVE-HE cell culture period. The gradual increase ofmyeloid lineage CD14⁺ cells appears to be associated with decrease inmegakaryocyte quality and platelet yield. In an effort to achieve higherand better yields of megakaryocyte progenitors, the c-myc gene wastargeted as an important regulatory gene during early megakaryopoiesiswith the aim of identifying novel megakaryocyte-promoting factors.

GSK1210151A (I-BET151) is an orally-available,imidazolonoquinoline-based inhibitor of the BET family of bromodomains.I-BET151 was shown to have profound efficacy against human and murineMLL-fusion leukemic cell lines, through the induction of early cellcycle arrest and apoptosis. The mode of action is partly due to theinhibition of transcription of key genes (BCL2, C-MYC, and CDK6) throughthe displacement of BRD3/4, PAFc, and SEC components through chromatin.

Although high dose i-BET-151 triggers massive apoptosis, treating cellsundergoing in vitro megakaryopoiesis with lower doses in the micromolarrange (or submicromolar range) resulted in increased numbers of MKprogenitors. FIG. 31 demonstrates, through quantitative mRNA analysis, adose-dependent inhibition of c-myc gene expression by i-BET-151. Incontrast, pro-MK gene GATA1 gene was up-regulated (FIG. 32) in responseto i-BET-151. Treatment with i-BET-151 also resulted in a dose-dependentdecrease in the number of CD14⁺ myeloid cells.

Thus, using the methods provided herein for in vitro generation ofiPS-derived (and ES-derived) megakaryocytes and platelets, it has beenfurther discovered that suppression of endogeneous c-myc gene expressionin cells undergoing megakaryopoiesis can change the balance of celldifferentiation in favor of the MK-lineage. In certain preferredembodiments, the megakaryocyte progenitors resulting from treatment withBET inhibitors or other c-myc inhibitors will, by Q-PCR analyses, showdown-regulation of MYC expression and up-regulation of GATA1 expressionrelative to untreated cells, i.e., down regulation of MYC by 10 percentor more, and more preferably down regulated by at least 20, 30, 40 oreven 50 percent relative to the megakaryocyte progenitors resulting inthe absence of treatment.

Example 11 Generation of Universal Platelets from Human Pluripotent StemCells

As described in the present application, human induced pluripotent stemcells and human embryonic stem cells provide a potentially replenishablesource for the production of transfusable platelets. This examplecontinues the description of methods to generate megakaryocytes (MKs)and functional platelets from iPSCs in a scalable manner underserum/feeder-free conditions. The method also permits thecryopreservation of MK progenitors, enabling a rapid “surge” capacitywhen large numbers of platelets are needed.Ultrastructural/morphological analyses show no major differences betweeniPSC-platelets and human blood platelets. iPSC-derived platelets formaggregates, lamellipodia and filopodia after activation, and circulatein macrophage-depleted animals and incorporate into developing mousethrombi in a manner identical to human platelets. By knocking-out theβ2-microglobulin gene, we have generated platelets that are negative forthe major histocompatibility antigens. The scalable generation ofHLA-ABC negative platelets from a renewable cell source represents thefirst step towards generating universal platelets for transfusion, aswell as a potential new strategy for the management of plateletrefractoriness.

Experimental Procedures

Reagents: STEMspan-ACF and STEMdiff-APEL Media, STEMspan MegakaryocyteExpansion Supplement (formerly CC-220), mTeSR1 medium, Dispase, andanimal protein-free defined cryopreservation medium CryoStor™ CS10 werepurchased from Stemcell Technologies (Vancouver, Canada). BMP4 was fromR&D systems and HumanZyme (Chicago, Ill.). All other cytokines wereobtained either from R&D systems or PeproTech (Rocky Hill, N.J.).Y-27632 was purchased from Stemgent (Cambridge, Mass.). Human CollagenIV was from Advanced BioMatrix (San Diego, Calif.). Matrigel andAntibodies for flow cytometry were obtained from BD Biosciences. I-BET151(GSK1210151A) was purchased from ChemieTek (Indianapolis, Ind.).StemPro Accutase was from Life Technologies (Grand Island, N.Y.).Heparin was purchased from Sigma (St. Louis, Mo.).

Human pluripotent stem cell cultures: Human iPSC line HA1-iPS wasreprogrammed by mRNA technology (Warren et al., 2012) and obtained fromAllele Biotechnology (San Diego, Calif.). Human iPS cell lines 19-9-11Tand 6-9-9T (WiCell, Madison, Wis.) were reprogrammed using episomalvectors technology. Human iPS line RHO8 was generated with retroviralvectors using fibroblasts derived from a RH-O blood type donor. HumanHDF-iPS-XA line (XA-iPS) was generated from human dermal foreskinfibroblasts using 6F Reprogramming Premix (Allele Biotechnology) underfeeder-free conditions in Pluriton Reprogramming Medium (Stemgent). hESClines MA09 and NED07 were derived using single blastomere technology(Klimanskaya et al., 2007). All human pluripotent stem cells used inthis study were cultured on a Matrigel-coated surface with mTeSR1medium. Confluent pluripotent stem cells were dissociated either withDispase (1 U/ml, Stemcell Technologies) or Cell Dissociation Buffer(CDB, Life Technologies). All pluripotent stem cells used in this studyhave normal karyotypes.

Generation of β2M^(KO) iPS cells: Derivation of β2M^(KO) iPS wasperformed by Cellectis Bioresearch (Paris, France) using transcriptionactivator-like effector nuclease (TALEN™) technology to disrupt the β2Mgene. Briefly, HA-iPS cells were transfected with a hsβ2M TALEN™targeting exon 2 and β2M^(KO) single cells were sorted by FACS. Afteramplification and clonal selection, the TALEN mediated deletion inestablished β2M negative iPS clones was validated by deep sequencing andthe lack of β2M expression was confirmed by FACS. The KO engineering ofHA-iPS was performed under feeder-free and xeno-free culture condition.

MK- and platelet-specific differentiation of pluripotent stem cells:Feeder-free iPS cells were dissociated with CDB and cell clumps werere-suspended in fresh mTeSR1 medium containing 10 μM ROCK inhibitor,Y27632. Cells were seeded into plates precoated with human collagen IV(5 μg/cm2 in 0.25% acetic acid) and allowed to attach at 37° C. in 5%CO2, 20% O₂ for 24 hours. Media was then changed to STEMspan-ACF+BMP4,VEGF and bFGF (50 ng/ml each) and cells were grown for 4 days underhypoxic condition (5% CO2, 5% O2) followed by 2 additional days at 5%CO2, 20% O2. On day 6, a representative culture was dissociated withAccutase and cell surface markers CD31, CD34, CD43, CD144 (VEcadherin),CD105 (Endoglin), CD184 (CXCR4) and CD309 (KDR) were analyzed by flowcytometry (Accuri C6).

To promote early MK progenitor production, cells were cultured inMK-Specific Progenitor Expansion medium (EXP-M) containing STEMdiff APELMedium+TPO (25 ng/ml), SCF (25 ng/ml), Flt-3 ligand (25 ng/ml), IL-3 (10ng/ml), IL-6 (10 ng/ml) and heparin (5 U/ml) for up to 7 days.Non-adherent MK Progenitors (MKPs) were constantly released into themedium and harvested for 4-5 consecutive days in EXP-M. Daily harvestswere cryo-preserved in animal protein-free defined cryopreservationCryoStor™ medium. Samples of MKPs were also subjected to flow cytometryfor analysis of CD41a, CD42b, CD31, CD34, CD43, CD13, CD235a, and CD14cell surface expression.

To induce MK maturation and platelet formation, freshly collected orthawed MKPs were cultured in MK maturation medium (MK-M) containingSTEMSpan-ACF+TPO, SCF, IL-6 and IL-9 (STEMspan Megakaryocyte ExpansionSupplement) and heparin (5 U/ml) in ultralow attachment plates(Corning). 5 μM of Y-27632 was added for the first 3 days of culture,and cells were incubated in 7% CO₂ at 39° C. Cell morphology and densitywere monitored daily and fresh medium was added to maintain 10⁶ cells/mlfor the first 4 days. The maturation of MKs from MKPs was monitoredusing flow cytometric analysis of CD41a, CD42b, and CD235a. Oncepro-platelet morphology was observed, medium samples were taken daily todetermine platelet yield using CD41a/CD42b double staining by flowcytometric analysis. Platelets were collected for 3-5 consecutive daysduring the peak of production. To extract platelets, large MKs wereremoved first using low speed centrifugation (50×g for 10 min), andproplatelets and small MKs were removed by further 10 min centrifugationat 300×g. The iPS-platelets in the supernatants were collected usinghigher g force (1000-2500×g) in the presence of PGE1 (1 μM). Thecollected iPS-platelet pellets were then subjected to furtherpurification using a BSA gradient centrifugation as described previously(Robert et al., 2012). Briefly, iPS-platelet pellets were re-suspendedwith 1× CGS buffer. Platelets were applied in the buffer on top of a BSAgradient with BSA concentration of (weight/volume) of 12, 10, 7, 5 and2%. The sample was centrifuged at 82×g for 15 min with no break, thenthe platelet-rich fraction was collected. iPS-platelets purified fromBSA gradient were stored or transported at room temperature in MK-M forfurther analyses.

Human blood platelet preparation and in vitro microscopiccharacterizations: Human blood was obtained by venipuncture from healthyvolunteers, as previously described (Thon et al., 2012). Collectionswere performed in accordance with ethics regulation with IRB approval,and informed consent was provided according to the Declaration ofHelsinki.

For electron microscopy, both human blood and iPS platelets were fixedwith 1.25% paraformaldehyde, 0.03% picric acid, 2.5% glutaraldehyde in0.1 μM cacodylate buffer and embedded in epoxy resin. Ultrathin sectionswere stained and examined with a Tecnai G2 Spirit BioTwin electronmicroscope (Hillsboro, Oreg.) as reported (Lu et al., 2011).

Differential interference contrast analysis was performed as previousreported (Lu et al., 2011). Briefly, samples were fixed in 4%formaldehyde and centrifuged onto poly-L-lysine (1 μg/mL)-coated slides.For microtubule components, samples were stained with an anti-β1-tubulinprimary antibody (Genemed Synthesis, San Antonio, Tex.); for actincomponents, samples were incubated with Alexa Fluor 568-conjugatedphalloidin (Invitrogen). To confirm cells were anucleate, samples wereincubated with Hoescht 33342 (Invitrogen). For granule localization,samples were incubated with antibodies against serotonin (EMDMillipore), thrombospondin 4 (Neomarkers), or platelet factor 4(Peptrotech) and treated with secondary antibodies, all conjugated toAlexa Fluor 488 (Invitrogen).

For contact activated spreading time lapse, iPS-platelets were pipettedinto chambers formed by mounting a glass coverslip onto a 10 mm petridish with a 1 cm hole. Platelets were permitted to contact glass bygravity sedimentation and spreading was captured at 5 second intervalsover a 5 minute period.

For size determination, platelets were individually thresholded fromβ1-tubulin-labeled samples and high-content diameter measurements wereperformed in ImageJ using the linescan and measurement functions.Analysis was confirmed by manual inspection of all samples, andimproperly thresholded cells were excluded from the analysis. More than100 cells were counted for each condition.

Platelet Kinetics in Live Animals: NOD/SCID mice (6-7 weeks old, male)were purchased from Jackson Laboratory (Bar Harbor, Me.). The Universityof Illinois Institutional Animal Care and Use Committee approved allanimal care and experimental procedures. Macrophages were depleted byintravenous injection of liposome-encapsulated clodronate as describedpreviously (Hu and Yang, 2012). Clodronate-liposomes were injected intomice through a tail vein at Day 0 (100 μl) and Day 2 (50 μl). At Day 3,human blood platelets and iPS-platelets were intravenously infused(1.5×10⁷ per mouse) into macrophage-depleted mice. Mouse blood, 30 μl,was collected through a jugular vein at different time points (10, 30,60, 120, 240, 360, and 480 minutes and 24 hours) and analyzed by flowcytometry using APC-conjugated anti-human CD41 and Dylight488-conjugated anti-mouse CD42c antibodies.

Real-time Fluorescence Intravital Microscopy: Intravital microscopy ofcremaster muscle arterioles was performed as previously described (Lu etal., 2011). NOD/SCID mice were depleted for macrophage, and thecremaster muscle arteriolar wall was injured by micropoint laserablation using a Micropoint Laser System (Photonics Instruments). Thedeveloping mouse platelet thrombus was visualized by infusion of Dylight649-conjugated anti-mouse CD42c antibodies (Emfret Analytics, 0.05 μg/gbody weight) and calcein AM-labeled human platelets and iPS-platelets(3×10⁶/mouse), were also infused with or without ReoPro (100 μg) intomice. Fluorescence and brightfield images were recorded using an OlympusBX61W microscope and a high speed camera (Hamamatsu C9300). Data werecollected for 5 min following vessel wall injury and analyzed usingSlidebook v5.5 (Intelligent Imaging Innovations).

Light Transmission Aggregometry and PAC-1 Activation Assay: HumaniPS-derived platelets or human peripheral and cord blood platelets werecounted on Sysmex Hematoanalyzer XE-2100D, spun down (15000 g, 15minutes), and resuspended in normal human plasma at the sameconcentration for light transmission aggregometry. 25 million plateletssuspended in plasma (270 μl) were placed in Chronolog cuvettes withChronolog stir bars, and stirring motion was set to 1000 rpm. Normalhuman plasma was used as blank. The baseline was set on Aggrolinksoftware, and agonist (either ADP or Thrombin in 30 μl) was added to afinal concentration of 20 μM or 1 U/mL, respectively (final reactionvolume=300 μl). Data was collected for 10 minutes, and aggregationpercentage was calculated with Aggrolink software. PAC-1 activationassay was performed as previously reported (Lu et al., 2011).

Expression of β2M and HLA-ABC on HA-iPS and β2M_(ko) iPS Derived MKs andPlatelets: Wild type HA-iPS and β2M^(KO) iPS cells were differentiatedinto MKs and platelets using method described above. Mature MKs fromboth lines were stained with FITC anti-human β2M antibody (Biolegend,Clone 2M2) and FITC anti-human HLA-A,B,C (Biolegend, clone W6/32), forcell surface expression using flow cytometry. FITC-isotype IgGs wereused as control. Platelets were obtained from HA-iPS and β2M^(KO) iPScells, and stained with PE anti-human CD42b (BD), APC anti-human CD41a(BD) together with either FITC anti-human β2M or FITC anti-human HLA19A,B,C antibodies. To exclude dead cells or other cellular fragments,only CD41a⁺CD42b⁺ platelet particles were counted for their β2M andHLA-ABC expression by flow cytometry. FITC isotype IgG staining was usedas control for β2M and HLA-ABC expression on iPS platelets. PAC1activation of platelets was performed as described previously.

Results

Efficient serum- and feeder-free direct differentiation of human iPScells to CD31⁺ hemogenic endothelial-like cells and MK progenitors:Feeder-free iPS cells were differentiated into hemogenicendothelial-like cells and hematopoietic progenitor intermediates priorto further differentiation into MKs and platelets. In the first six daysof differentiation, we observed dramatic morphological changes: compactcolonies (Day 0) stretched out and became loose colonies (Day 3), whichgrew out and formed sheets with a complex morphology by Day 6. Cellsurface marker analysis on Day 6 showed that 58.5% (±3.74%) of attachedcells were CD31⁺. Approximately 34.43% of these cells were CD34⁺ andless than 10% of them were CD43⁺, indicating an early stage ofhematopoietic commitment (Vodyanik et al., 2006). Further analysesdemonstrated that between 30-60% of the attached cells also expressedCD144 (VE-Cadherin), CD105, CXCR4 and KDR. As CD31(PECAM-1) has beenshown to be an important marker for hemogenic endothelium (Oberlin etal., 2002), we further determined the expression of several surfacemarkers that are also expressed on hemogenic endothelium on both ourCD31⁺ and CD31⁻ cells. FACS analyses demonstrated that about 67.5% and63.1% of CD31⁺ cells expressed CD105 and VE-Cadherin, respectively;whereas only about 10.9% and 5.8% of CD31⁻ cells were positive for CD105and VE-cadherin. Hematopoietic colony forming assays showed that CD31⁺cells possess robust hematopoietic potential with the ewhereas CD31⁻cells developed very limited numbers of hematopoietic colonies. Afterplating on a fibronectin coated surface, CD31⁺ cells formed a monolayerwith characteristics of endothelium morphology, expressed high level ofvon Willibrand Factor and took up Ac-LDL. These results demonstrate thatCD31⁺ hemogenic endothelial-like cells can be efficiently generated bydirect differentiation of feeder-free iPS cells under definedconditions.

Six days after initial differentiation, culture medium was replaced byexpansion medium EXP-M. From D6+1 to D6+4, large numbers of roundedcells grew out of the attached cells. The majority of these floatingcells were CD31⁺CD34⁺CD43⁺, indicating their commitment toward thehematopoietic lineage (Vodyanik et al., 2006). These floating cells werealso CD41a⁺CD13⁺CD14⁻, and expression of the MK specific marker CD42bwas variable (9.5-37%). We therefore defined these cells asmegakaryocyte progenitors (MKP) by cell surface marker expression(CD31⁺CD34⁺CD43⁺CD41a⁺CD13⁺CD14⁻CD42b^(+/−)). However, we also observedthat CD14⁺ myeloid cells (which are almost 100% CD42b⁻) increased from<1% to about 38% from Day6+1 to Day6+7, indicating the expansion rate ofmyeloid cells surpassed that of MKPs during this time period. In fivelarge-scale experiments, we generated a total of 2.06×10⁹ MKPs from1.26×10⁸ iPS cells, an average of >16 MKPs per single iPS cell input.

Down-regulation of MYC promotes MKP formation and prohibits myeloid cellexpansion: Previous studies suggest that the c-myc gene may be involvedin megakaryopoiesis (Chanprasert et al., 2006; Guo et al., 2009;Thompson et al., 1996). As also described above in Example 10, wetherefore determined whether suppression of c-myc expression wouldenhance MK lineage differentiation/expansion and simultaneously prohibitmyeloid and other lineage differentiation/expansion. MKP Day 6+3cultures were treated with non-cytotoxic doses of a c-myc inhibitor,iBET151 (0.1 and 0.25 μM) for 24 hours and then harvested for analysis.Q-PCR analyses showed a dose-dependent downregulation of MYC andup-regulation of GATA1 in MKPs treated with iBET151. Supplementation ofiBET151 at 0.1 and 0.25 μM resulted in ≈2 and 4 fold increases of MKPoutputs, respectively, as compared to untreated controls. Results alsoshowed that addition of iBET151 from D6+3 to D6+4 inhibited theformation of CD14₊ myeloid cells in a dose-dependent manner. CD14₊myeloid cells were decreased from about 12% in control cultures to lessthan 5% and 3% in cultures treated with iBET151 at 0.1 and 0.25 μM,respectively. These results suggest that the c-myc gene not only plays akey role in early megakaryopoiesis, which is consistent with previousreports (Takayama et al., 2010), but that it may be involved in thedifferentiation and expansion of other hematopoietic lineages.

Feeder-free generation of platelets from iPS-MKs: We previously foundthat 40-50% CD41a⁺/CD42b⁺ double positive platelets were generated fromMKs with stroma/feeder co-culture whereas this amount decreased to <15%when feeder-free conditions were used (Lu et al., 2011). To overcome therequirement for feeders and improve production of CD41a⁺/CD42b⁺ doublepositive MKs and platelets, we tested several conditions with differentbasal medium and cytokine/growth factor combinations. Our results showedthat a combination of STEMspan-ACF medium with STEMspan MegakaryocyteExpansion Supplement (StemCell Technologies) as the core components ofMK maturation medium was the best media and cytokine mixture tested foriPS-MKP maturation and subsequent platelet production. Culturing of MKPsin this medium for 4 days resulted in a rapid increase of CD41a⁺CD42b⁺double positive matured MK population from ≈20% to >80%, and the almostcomplete elimination of CD235A⁺ erythroid cells. These results indicatethat this MK maturation medium efficiently steers cells towards MKlineage development while suppressing the expansion of erythroid cellseven though these cell types share a common bipotential progenitor(Klimchenko et al., 2009).

Although results vary among different cell lines, large MKs anddistinctive pro-platelet structures were present in 3-4 days of MKcultures. No difference was observed between MKs derived from iPS andhES cells in forming proplatelets. To determine if proplatelets in thecultures reflect the generation of platelets, we examined CD41a/CD42bexpression on iPS-platelets purified by BSA gradient segregation (Robertet al., 2012). Using human blood platelets to establish proper sizegating, we observed that more than 70% of both iPS- and hES-plateletsgenerated under feeder and serum-free conditions expressed both CD41aand CD42b, which is similar to human blood platelets (82%). To ourknowledge, this is the first time that platelets with such high purityhave been generated in vitro, even under conditions with stromal cellsand serum. We found that the quality of platelets (CD41a⁺/CD42b⁺expression) generated from iPS-MKs was inversely correlated with thepercentage of CD14⁺ myeloid cells in the starting MK cultures,indicating that a pure MK population is critical for the generation offunctional platelets in vitro. The kinetics of platelet production wasalso monitored, and both the purity and quantity of platelets in cultureincreased gradually from day 4 and peaked at day 6.

Proteases such as MMPs are known to be involved in shedding of CD42b(GPIbα), which is the receptor for vWF and mediates initial plateletreactions to wounds. Loss of CD42b is closely associated with a declinein platelet quality. A broad-spectrum MMP inhibitor, GM6001 (GM), isreported to inhibit CD42b shedding from platelets (Nishikii et al.,2008; Robert et al., 2011). Consistent with our previous study (Lu etal., 2011), the addition of GM in MK culture protected CD42b onplatelets from shedding, resulted in an increase of CD41b⁺/CD42b⁺platelets from 45% to 59%. However, FACS analysis of GM-treatedplatelets showed a less homogeneous profile. In a systemic comparativestudy using several specific MMP inhibitors, we identified anMMP8-specific inhibitor (MMP8-I) with superior shedding protection thanGM. Addition of e importantly, MMP8-I treated platelets displayed morehomogeneous FACS profile. Total number of CD41a⁺CD42b⁺ plateletsobtained from MMP8-I e (˜46% increase vs control), However, nosignificant synergistic effect was observed when combining MMP8-I andGM.

Mild hyperthermia (39° C. instead of 37° C.) reportedly improves MKdifferentiation/maturation and platelet generation from umbilical cordblood CD34⁺ cells (Pineault et al., 2008; Proulx et al., 2004). However,it is unclear whether mild hyperthermia will be beneficial toiPS-derived MK differentiation/maturation and platelet production. Usingthe above described method, we found that mild hyperthermia (39° C.)improved both the purity (percentage of CD41a⁺/CD42b⁺) and the yield ofplatelets from iPS-MKs during the peak platelet production from day 4 to7 of MK cultures

Ultrastructural and functional characterization of iPS-platelets invitro: Thin section electron micrography showed that iPS-platelets wereultrastructurally similar to circulating human platelets. TheiPS-platelets have a discoid shape, display a smooth contour and containa normal distribution of the open canalicular system as well as α- anddense granules and other organelles, which are indistinguishable fromfeatures of human blood platelets. Immunofluorescence micrographs showedthat iPS-platelets were anucleate but on average, slightly larger thancirculating human blood platelets (2.38±0.85 μm versus 2.27±0.49 μm,n>100). However, size distribution of iPS-platelets was similar to humanblood platelets, and these platelets have both normal tubulincytoskeleton and filamentous actin relative to circulating humanplatelets, and strongly express thrombospondin 4 and platelet factor 4(α-granule markers). In summary, similar to hES-platelets generated withstromal cells (Lu et al., 2011), iPS-platelets displayed all of theultrastructural and morphological criteria that are characteristic ofblood platelets.

To determine whether iPS-platelets generated under this stroma-freecondition can be activated, we performed a binding assay using the PAC-1monoclonal antibody, which only binds to the activated conformation ofαIIbβ3 integrin. In response to thrombin treatment, iPSplatelets showedapproximately a 6-fold increase in PAC-1 binding as compared to restingcontrols, which is similar to hES-platelets produced with stromal cells,but weaker than human blood platelets as reported previously (Lu et al.,2011; Takayama et al., 2008). Live cell microscopy revealed thatiPS-platelets spread on a glass surface and formed lamellipodia andfilopodia with membrane ruffling after stimulation; they also spread outand tethered to each other, mimicking the early stage of aggregation,which are typically observed in blood platelets in response toactivation stimulation.

Light transmission aggregometry (LTA) is the most common method used inclinical and research laboratories to assess platelet function (Panzerand Jilma, 2011). LTA has never been performed with platelets generatedfrom human PSCs in vitro, presumably due to its requirement for a largequantity of fresh platelets. Human peripheral blood (PB) platelets(2.5×10⁷) resuspended in human plasma reached ≈80% aggregation 6 minafter exposed to 1 U/ml thrombin. Similar numbers of iPS-plateletsresponded to 1 U/ml of thrombin in forming aggregates, but the processwas slower and aggregation was weaker as compared to PB platelets: only<30% of aggregation was observed 6 min after thrombin stimulation. HumanCB platelets similarly showed weak aggregation (≈10%) under the sameconditions, which is consistent with previous observations that CBplatelets showed a weaker aggregation compared to PB platelets in LTAassay (Israels, 2013; Israels and Rand, 2013; Israels et al., 2003).Similar results were observed after exposed PB and CB platelets, andiPS-platelets to 20 μM ADP, the most common agonist used for plateletaggregation studies. However, our results are a significant improvementover in vitro CD34⁺ cell-derived platelets, which showed no aggregationat all by LTA (Robert et al., 2011).

iPS-platelets generated under completely serum- and feeder-freeconditions are functional in vivo: Previous studies demonstrated thatmouse macrophages play a major role in rejecting human platelets (Hu andYang, 2012). To investigate the kinetics and in vivo functionality ofiPS-platelets, NOD/SCID mice were treated without or withliposome-encapsulated clodronate as described previously (Hu and Yang,2012), and 1.5×10⁷ and 5×10⁸ blood human platelets were intravenouslyinfused into these animals. Mouse blood samples were collected atdifferent time points and analyzed by flow cytometry with antibodiesspecifically against human CD41 and CD42 antigens. We observed thathuman platelets were removed within 10 min from the circulation ofliposome treated control mice even with the infusion of 5×10⁸ humanplatelets, whereas human platelets circulated for at least 8 hours inmacrophage-depleted mice. Therefore, NOD/SCID mice were pre-treated withliposome-encapsulated clodronate to deplete macrophage, and 1.5×10⁷iPS-platelets/mouse were infused intravenously and mouse blood sampleswere analyzed for human platelets at different times. HumaniPS-platelets, like human blood platelets, circulated for at least 8hours in macrophage-depleted NOD/SCID mice with a Tmax of 0.5-1 hour,however no circulating human blood platelets or iPS-platelets weredetected 24 hours after infusion.

In the examples above we taught that hES-platelets, like human bloodplatelets, incorporated into the developing mouse thrombus at the siteof laser-induced arteriolar injury in live mice (Lu et al., 2011). Toinvestigate whether iPS-platelets are functional in vivo, similarexperiments were performed in macrophage-depleted NOD/SCID mice. Likehuman blood platelets and hES-platelets, iPS-platelets incorporated intothe growing platelet thrombus with an average number of 9.0±1.8platelets per thrombus, which was indistinguishable from human bloodplatelets. Infusion of human blood platelets or iPS-platelets did notalter the kinetics of mouse platelet thrombus formation at the site ofarteriolar injury (Tmax=85-105 seconds). To examine whether theincorporation of iPS-platelets into the developing thrombus is mediatedby αIIbβ3 integrin, ReoPro (100 μg), a specific inhibitor for humanαIIbβ3 integrin, was infused into the same mouse treated with humanplatelets or iPS-platelets. Treatment with ReoPro markedly abolishedhuman platelet or iPS-platelet binding to the growing thrombus at theinjury sites, whereas ReoPro did not affect the number of circulatingiPS-platelets and the formation of mouse platelet thrombi. These resultssuggest that iPS-platelets, like human blood platelets andhES-platelets, are functional in vivo.

Generation of HLA major negative MKs and platelets from β2M_(ko) iPScells: MKs and platelets were generated from β2M^(KO) iPS cells and theexpression of both β2M and HLA-ABC was measured with flow cytometry incomparison to those generated from parental HA-iPS cells. The β2M^(KO)iPS cells displayed a normal MK lineage specific differentiationcapability, same as their parental HA-iPS cells. MKs derived from HA-iPScells, as expected, expressed both β2M and HLA-ABC, while the expressionof both antigens in β2M^(KO) iPS MKs was undetectable. Similarly, flowcytometry analyses confirmed that platelets generated from ≢2M^(KO) iPScells did not express HLA-ABC, whereas platelets derived from parentaliPS cells were HLA-ABC positive. Both HLA-ABC positive and negativeplatelets possessed similar activation capacity as shown by PAC-1binding activity after thrombin treatment (47.1% vs 53.8%).

1. A pharmaceutical preparation that is suitable for use in a humanpatient comprising at least 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴platelets, wherein the preparation is substantially free of leukocytesand wherein substantially all of the platelets are functional. 2.(canceled)
 3. The pharmaceutical preparation of claim 1, wherein theplatelets have one or more of the following attributes: a mean plateletvolume range of 9.7-12.8 fL; and/or a unimodal distribution of size inthe preparation; and/or a log normal platelet volume distributionwherein one standard deviation is less than 2 μm³ (preferably less than1.5 μm³, 1 μm³ or even 0.5 μm³); and/or positive for CD41a and/or CD42b;and/or at least 50%, 60%, 70%, 80% or 90% of the platelets arefunctional for at least 2, 3 or 4 days after storage at roomtemperature. 4.-7. (canceled)
 8. A composition comprising at least 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴⁻ megakaryocyte lineage specificprogenitors (MLPs).
 9. The composition of claim 8, wherein thecomposition is cryopreserved. 10.-14. (canceled)
 15. A method forproducing platelets from megakaryocytes comprising the steps of: a)providing a non-adherent culture of megakaryocytes; b) contacting themegakaryocytes with hematopoietic expansion medium and (1) TPO or a TPOagonist, SCF, IL-6 and IL-9; or (2) TPO or a TPO agonist, SCF, andIL-11; or (3) TPO or a TPO agonist to cause the formation ofpro-platelets in culture, wherein the pro-platelets release platelets,and c) isolating the platelets.
 16. The method of claim 15, wherein saidTPO agonist comprises one or more of: ADP, epinephrine, thrombin,collagen, TPO-R agonists, TPO mimetics, second-generation thrombopoieticagents, romiplostim, eltrombopag (SB497115, Promacta), recombinant humanthrombopoietin (TPO), pegylated recombinant human megakaryocyte growthand development factor (PEG-rHuMGDF), Fab 59, AMG 531, Peg-TPOmp, TPOnonpeptide mimetics, AKR-501, monoclonal TPO agonist antibodies,polyclonal TPO agonist antibodies, TPO minibodies, VB22B sc(Fv)2, domainsubclass-converted TPO agonist antibodies, MA01G4G344, recombinant humanthrombopoietins, recombinant TPO fusion proteins, or TPO nonpeptidemimetics.
 17. (canceled)
 18. The method of claim 15, wherein thenon-adherent culture of megakaryocytes is a feeder-free culture.
 19. Themethod of claim 15, wherein the culture in step (b) is in a mediumcomprising one or more of: Stem Cell Factor (SCF) at 0.5-100 ng/ml,Thrombopoietin (TPO) at 10-100 ng/ml, and Interleukin-11 (IL-11) at10-100 ng/ml, at least one ROCK inhibitor, and/or Heparin at 2.5-25Units/ml; or in a medium comprising one or more of: TPO at 10-100 ng/ml,SCF at 0.5-100 ng/ml, IL-6 at 5-25 ng/ml, IL-9 at 5-25 ng/ml, at leastone ROCK inhibitor, and/or Heparin at 2.5-25 units/ml. 20.-22.(canceled)
 23. The method of claim 15, further comprising subjecting themegakaryocytes to a shearing force. 24.-30. (canceled)
 31. The method ofclaim 15, wherein the megakaryocytes are cultured in the presence of anexogenously added protease inhibitor or an exogenously added MMPinhibitor. 32.-34. (canceled)
 35. The method of claim 15, wherein themegakaryocytes are cultured at a temperature greater than 37° C. andequal to or less than 40° C.
 36. The method of claim 15, wherein themegakaryocytes are generated by steps comprising: (a) culturingpluripotent stem cells to form hemogenic endothelial cells (PVE-HE); (b)culturing the hemogenic endothelial cells to form MLPs; and (c)culturing the MLPs to form megakaryocytes.
 37. The method of claim 36,wherein a BET inhibitor is added to the culture in step (b). 38.-39.(canceled)
 40. The method of claim 36, wherein the hemogenic endothelialcells are derived without embryoid body formation. 41.-43. (canceled)44. The method of claim 36, wherein said hemogenic endothelial cells aredifferentiated from the pluripotent stem cells under low oxygenconditions comprising 1% to 10% oxygen, 2% to 8% oxygen, 3% to 7%oxygen, 4% to 6% oxygen, or about 5% oxygen.
 45. (canceled)
 46. Apharmaceutical preparation comprising platelets produced by the methodof claim
 15. 47.-50. (canceled)
 51. A method of treating a patient inneed of platelet transfusion, comprising administering the compositionof claim 1 to said patient.
 52. (canceled)
 53. A method for producingplatelets from megakaryocytes or MLPs comprising culturing anon-adherent population of megakaryocytes or MLPs under shear forceconditions in the presence of a protease inhibitor, and harvestingplatelets from the culture. 54.-68. (canceled)
 69. A method forproducing platelets from megakaryocytes or MPLs comprising culturing anon-adherent population of megakaryocytes or MPLs derived from iPS cellsor ES cells at a temperature greater than 37° C. and equal to or lessthan 40° C., and harvesting and optionally isolating platelets from theculture. 70.-73. (canceled)
 74. A method of treating a patient in needof platelet transfusion, comprising administering the composition ofclaim 46 to said patient.